This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.
Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.
Oncolytic viruses (OVs) are being developed as anti-cancer therapeutics that specifically replicate within and kill tumor cells while leaving healthy tissues intact. It has now become common understanding that oncolytic virotherapy (OVT), in most cases, does not rely solely on complete tumor lysis by efficient replication and spreading of the virus, but requires additional mechanisms of action for treatment success, including vascular and stromal targeting and, importantly, immune stimulation1,2,3,4. While many early OV studies used unmodified viruses, current research has profited from an improved biological understanding, virus biobanks that potentially contain novel OVs, and the possibilities offered by genetic engineering in order to create advanced OV platforms5,6,7.
Given the recent success of immunotherapy, immunomodulatory transgenes are of particular interest regarding the genetic engineering of OVs. Targeted expression of such gene products by OV-infected tumor cells reduces toxicity compared to systemic administration. Targeting is achieved either by using viruses with inherent oncoselectivity or by modifying viral tropism8. Local immunomodulation enhances the multi-faceted anti-tumor mechanisms of OVT. Furthermore, this strategy is instrumental in interrogating the interplay between viruses, tumor cells, and the host immune system. To this end, this protocol provides an applicable and adjustable workflow to design, clone, rescue, propagate, and validate oncolytic paramyxovirus (specifically measles virus) vectors encoding such transgenes.
Modulation of the immune response can be achieved by a wide variety of transgene products targeting different steps of the cancer-immunity cycle9, including enhancing tumor antigen recognition [e.g., tumor-associated antigens (TAAs) or inducers of major histocompatibility complex (MHC) class I molecules] over supporting dendritic cell maturation for efficient antigen presentation (cytokines); recruiting and activating desired immune cells such as cytotoxic and helper T cells [chemokines, bispecific T cell engagers (BTEs)]; targeting suppressive cells such as regulatory T cells, myeloid-derived suppressor cells, tumor-associated macrophages, and cancer-associated fibroblasts (antibodies, BTEs, cytokines); and preventing effector cell inhibition and exhaustion (checkpoint inhibitors). Thus, a plethora of biological agents is available. Evaluation of such virus-encoded immunomodulators regarding therapeutic efficacy and possible synergies as well as understanding of respective mechanisms is necessary to improve cancer therapy.
Negative sense single-stranded RNA viruses of the Paramyxoviridae family are characterized by several features conducive to their use as oncolytic vectors. These include a natural oncotropism, large genomic capacity for transgenes (more than 5 kb)10,11, efficient spreading including syncytia formation, and high immunogenicity12. Therefore, OV platforms based on canine distemper virus13, mumps virus14, Newcastle disease virus15, Sendai virus16,17, simian virus 518, and Tupaia paramyxovirus19 have been developed. Most prominently, live attenuated measles virus vaccine strains (MV) have progressed in preclinical and clinical development20,21. These virus strains have been used for decades for routine immunization with an excellent safety record22. Moreover, there is no risk for insertional mutagenesis due to the strictly cytosolic replication of paramyxoviruses. A versatile reverse genetics system based on anti-genomic cDNA which allows for insertion of transgenes into additional transcription units (ATUs) is available11,23,24. MV vectors encoding sodium-iodide symporter (MV-NIS) for imaging and radiotherapy or soluble carcinoembryonic antigen (MV-CEA) as a surrogate marker for viral gene expression are currently being evaluated in clinical trials (NCT02962167, NCT02068794, NCT02192775, NCT01846091, NCT02364713, NCT00450814, NCT02700230, NCT03456908, NCT00408590, and NCT00408590). Safe administration has been confirmed and cases of anti-tumor efficacy have been reported in previous studies25,26,27,28,29,30 (reviewed by Msaouel et al.31), paving the way for additional oncolytic measles viruses that have been developed and tested preclinically. MV encoding immunomodulatory molecules targeting diverse steps of the cancer-immunity cycle have been shown to delay tumor growth and/or prolong survival in mice, with evidence for immune-mediated efficacy and long-term protective immune memory in syngeneic mouse models. Vector-encoded transgenes include granulocyte-macrophage colony stimulating factor (GM-CSF)32,33, H. pylori neutrophil-activating protein34, immune checkpoint inhibitors35, interleukin-12 (IL-12)36, TAAs37, and BTEs38, which cross-link a tumor surface antigen with CD3 and thus induce anti-tumor activity by polyclonal T cells, irrespective of T cell receptor specificity and co-stimulation (Figure 1). The promising preclinical results obtained for these constructs demand further translational efforts.
Talimogene laherparepvec (T-VEC), a type I herpes simplex virus encoding GM-CSF, is the only oncolytic therapeutic approved by the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA). The phase III study leading to approvals in late 2015 has not only shown efficacy at the site of intra-tumoral injection, but also abscopal effects (i.e., remissions of non-injected lesions) in advanced melanoma39. T-VEC has since entered additional trials for application in other tumor entities (e.g., non-melanoma skin cancer, NCT03458117; pancreatic cancer, NCT03086642) and for evaluation of combination therapies, especially with immune checkpoint inhibitors (NCT02978625, NCT03256344, NCT02509507, NCT02263508, NCT02965716, NCT02626000, NCT03069378, NCT01740297, and Ribas et al.40).
This demonstrates not only the potential of oncolytic immunotherapy but also the need for further research to identify superior combinations of OVT and immunomodulation. Rational design of additional vectors and their development for preclinical testing is key to this undertaking. This will also advance understanding of underlying mechanisms and has implications for the progression towards more personalized cancer treatment. To this end, this publication presents the methodology for the modification and development of paramyxoviruses for targeted cancer immunotherapy and, more specifically, of oncolytic measles viruses encoding T cell-engaging antibodies (Figure 2).
NOTE: [O], [P], and [M] indicate subsections applicable to: OVs in general, (most) paramyxoviruses, or MV only, respectively. [B] indicates sections specific for BTE transgenes.
1 Cloning of Immunomodulator-encoding Transgenes into Measles Virus Vectors
2. Rescuing Recombinant Measles Virus Particles Encoding Immunomodulators
3. Determining Replicative and Cytotoxic Capacities of Viral Vectors Encoding Immunomodulators
4. Analyzing Activity of Virus-encoded Immunomodulators Secreted from Infected Cells
Figure 1 illustrates the mechanism of action of oncolytic measles viruses encoding bispecific T cell engagers. A flowchart depicting the workflow of this protocol is presented in Figure 2. Figure 3 shows an example of a modified oncolytic measles virus genome. This provides a visual representation of the specific changes applied to the measles virus anti-genome and particular features of inserted transgenes. Typical measles virus-induced syncytia are depicted in Figure 4. Note the high cell density at the timepoint of harvesting the rescue (A, B), indicating that the cell number may be reduced when repeating the experiment. Incubation temperatures and -times may be optimized for passaging on Vero cells (C, D) to achieve improved spread of syncytia across the plate. Figure 5 represents the outcome of a typical titration assay. In Figure 6, one-step growth curves (A) and relative cell viability (B) after inoculation with unmodified (MV) and BTE-encoding oncolytic measles viruses (MV-H-mCD3xhCD20) are shown. While growth curves of the compared vectors on Vero cells appear similar, lytic activity of the transgene-encoding virus lags behind in the murine tumor cell line. Flow cytometry data of target antigen-expressing cells incubated with BTEs at five different dilutions is provided in Figure 7, indicating BTE binding by cells in a concentration-dependent manner. Figure 8 represents an exemplary immunoblot after magnetic pulldown of BTE-associated cells. Pulldown with non-targeting BTEs (n1, n2) did not yield detectable amounts of cells, whereas bands in the elution fraction, indicative of bound cells, were observed for targeting BTE samples (t1, t2). BTE-mediated, target antigen-specific and concentration-dependent cytotoxicity of murine T cells is indicated by a representative LDH release assay in Figure 9.
Figure 1: Mechanism of action of oncolytic measles viruses encoding a bispecific T cell engager (MV-BTE). Infection of a tumor cell (infected: grey, uninfected: light blue) with MV-BTE is followed by viral replication and spread throughout the tumor, resulting in tumor cell lysis and immune stimulation. Simultaneously, BTEs [consisting of two single chains derived from antibodies targeting CD3 on T cells (yellow) and a tumor surface antigen (blue)] are produced and secreted locally by virus-infected cells. BTE-mediated cross-linking with tumor cells induces activation of resting, polyclonal T cells, resulting in further tumor cell killing. Please click here to view a larger version of this figure.
Figure 2: Flowchart describing the workflow of designing, generating, and evaluating novel viral vectors encoding immunomodulatory transgenes. Steps 1 to 4 reflect the respective sections of the protocol. Bullet points indicate relevant considerations and substeps. Due to the empirical nature of the procedure, adjustments or refinements can become necessary at any step of the workflow. In addition, experimental findings can lead to new hypotheses and inspire development of additional vectors or combination treatments. Please click here to view a larger version of this figure.
Figure 3: Schematic representation of a measles virus genome. Enhanced green fluorescent protein (eGFP) is encoded in an additional transcription unit downstream of the leader (ld) sequence. N, P, M, F, H, and L designate genes encoding the measles virus structural proteins nucleoprotein, P protein, matrix protein, fusion protein, hemagglutinin protein, and large protein (polymerase), respectively, which are differentially expressed as visualized above. A bispecific T cell engager (BTE) transgene is inserted into an additional transcription unit (ATU) downstream of the H open reading frame. The transgene encodes an Igκ leader peptide for efficient secretion as well as influenza hemagglutinin (HA) and hexa-histidine (His) protein tags for detection and purification. Genes coding for variable heavy (VH) and light chain (VL) domains of antibodies targeting CD3 and CD20, respectively, are connected via peptide linker sequences containing glycine (G) and serine (S) residues. The transgene sequence is preceded by a Kozak sequence. Downstream of the coding region, additional nucleotides have been included exemplarily to comply with the rule of six. The insert cassette is flanked by two restriction sites enabling insertion into the respective ATU. Please click here to view a larger version of this figure.
Figure 4: Measles virus-induced syncytia. (A, B) Vero cells were transfected with cDNA for rescue of recombinant measles viruses encoding enhanced green fluorescent protein (eGFP) and a bispecific T cell engager (BTE) targeting murine CD3 and human CD20. Presence of a fluorescent syncytium (white arrows) indicates successful rescue of infectious virus. (C, D) Measles viruses were harvested after rescue and propagated on Vero cells (passage 1). Large syncytia have formed and are already starting to burst (yellow arrows). Images were acquired by phase contrast (B, D) and fluorescence microscopy after excitation for 80 ms (A) and 100 ms (C). Please click here to view a larger version of this figure.
Figure 5: Representative result of a titration assay. Titration of a third-passage batch of MV-H-mCD3xhCD20, a BTE-encoding oncolytic measles virus, on Vero cells. A 10-fold serial dilution was performed on a 96-well plate, starting with 10 µL of virus suspension in each well of column 1.No syncytia were visible in column 7, as indicated by zeros. For the next lowest dilution of virus suspension in column 6, an average of six syncytia per well was observed, resulting in a final titer of approximately 6 x 107 cell infectious units (ciu) per mL virus suspension. Please click here to view a larger version of this figure.
Figure 6: Replication kinetics and lytic activity of recombinant measles viruses. (A) One-step growth curves were generated after infection of Vero cells with unmodified oncolytic measles virus (MV) or measles virus encoding a bispecific T cell engager (MV-H-mCD3xhCD20) at an MOI of 1. Titers of viral progeny were evaluated at designated timepoints after infection, indicating similar virus replication kinetics. Means and standard deviations of eight samples (four technical replicates each of two biological replicates per timepoint) are shown. (B) Cell viability was assessed on MC38 murine colorectal carcinoma cells stably expressing human carcinoembryonic antigen and the MV receptor CD46 (MC38-CEA-CD46) after inoculation with medium only (mock) or indicated viruses at an MOI of 1. XTT cell viability assay was performed at indicated timepoints. Reduction in cell viability was observed at earlier timepoints for the unmodified vector. Values of over 100%, as calculated for MV-H-mCD3xhCD20 at the 12 h timepoint, are frequently observed shortly after infection, which may be due to cellular stress or cell-derived factors present in the virus suspension. Mean values plus standard deviations of three technical replicates are shown for each timepoint. Please click here to view a larger version of this figure.
Figure 7: Target cell binding by bispecific T cell engagers (BTEs) expressed from measles virus-infected cells. Human mantle cell lymphoma cells endogenously expressing CD20 (Granta-519) were incubated with human immunoglobulin G to block Fc receptors, followed by incubation with 2, 4, 6, 8, or 10 µL (A) of purified mCD3xhCD20 BTE solution, respectively. BTE-bound cells were detected using phycoerythrin (PE)-conjugated antibody targeting the BTE-associated HA-tag. Percentages of stained cells correlated with BTE concentrations. (B) Controls for selectivity and specificity of binding. Shown here are controls from an independent experiment, including one sample each of cells not incubated with BTE but with the BTE-targeting antibody only (control for unspecific binding of antibody to cells) or with a BTE that is known to bind to the cells of interest, followed by incubation with either the BTE-targeting antibody (positive control) or an isotype antibody. If available, isogenic cells not expressing the target antigen of choice may be used to further verify binding selectivity. Please click here to view a larger version of this figure.
Figure 8: Magnetic pulldown of human peripheral blood mononuclear cells (PBMCs) bound by measles virus-encoded BTEs. Cells were incubated with two different batches of human T cell-targeting (t1, t2) or non-targeting control BTEs (n1, n2), respectively. BTE-bound cells were retained on columns using magnetic beads, pelleted, and lysed. β-actin in lysates was detected by immunoblotting. Intensities of bands in the elution samples indicate relative BTE-cell binding. Flow-through specimens confirm presence of cells in all samples. Please click here to view a larger version of this figure.
Figure 9: Cytotoxic activity of measles virus-encoded BTEs. Target tumor cells (5 x 10³ B16-CD20-CD46 cells per well) were incubated with murine T cells at a ratio of 1:50. BTEs previously purified from the supernatant of MV-H-mCD3xhCD20-infected cells were added at indicated concentrations. Relative lysis of target cells was assessed by LDH release assay after 48 h. Cells lacking the BTE target antigen (B16-CD46) served as reference to evaluate antigen-specific cytotoxicity. Means plus standard deviations of three technical replicates per sample are shown. Target antigen-expressing cells were specifically lysed in a BTE concentration-dependent manner. Purity of the BTE product and target antigen expression levels influence cell killing. In the present example, 15% specific cell killing was achieved at a relatively high BTE concentration of 1 µg/mL. This is a typical value for such an experimental setup with long co-incubation times and suboptimal T cell culture conditions. In other settings, up to 60% specific killing was achieved using BTEs purified from MV-infected supernatants, reaching a plateau at BTE concentrations of 100 ng/mL and higher38. This indicates that, counter-intuitively, the limit of this assay is less than 100% specific killing, which can be explained by different growth kinetics in Tmax controls compared to co-culture samples. Please click here to view a larger version of this figure.
Oncolytic immunotherapy (i.e., OVT in combination with immunomodulation) holds great promise for cancer treatment, demanding further development and optimization of oncolytic viruses encoding immunomodulatory proteins. This protocol describes methods to generate and validate such vectors for subsequent testing in relevant preclinical models and potential future clinical translation into novel anti-cancer therapeutics.
Numerous different oncolytic virus platforms with distinct advantages are available63. In addition to cancer specificity, vector safety, requirements for manufacturing, efficacy in tumor cell lysis, and induction of immune responses, cloning capacity is key for successful vectorization of immunomodulators using a specific oncolytic. Unfortunately, direct comparisons of different OVs are currently lacking and should be pursued in order to identify optimal treatment options for individual patients. This can be facilitated by promoting the rational development and testing of novel transgene-encoding vectors, as exemplified here for MV-BTE. Given the beneficial properties of MV (i.e., oncotropism, safety, fusogenicity, immunogenicity, feasibility of genetic modification) this protocol focuses on this oncolytic vector, which can be generalized for other OV, especially paramyxoviruses.
For a rational choice of potentially relevant immunomodulators as candidate transgenes (step 1.1.1), a thorough understanding of the cancer-immunity cycle is essential, making systematic literature research indispensable. In addition, large-scale screens, though costly, can prove valuable to identify novel targets (e.g., see Patel et al.41).
For design of recombinant OVs, desired expression profiles should be considered. In the case of RNA viruses, gene expression is controlled on the RNA level by the respective polymerase. When designing transgene cassettes for insertion into MV genomes (step 1.1.2), avoiding sequences that resemble MV polymerase gene start/stop signals and RNA editing sites and following the rule of six is crucial for successful vector development. Moreover, paramyxovirus gene expression levels correspond to positioning within the genome, resulting from an expression gradient43. In general, positioning of the transgene in an upstream position increases expression at the expense of reduced replication. However, these parameters also depend on the size, structure, and sequence of the respective transgene. Consequently, a limitation of the methodology described in this protocol is the need for empirical testing of novel vector designs. In specific cases, adjustments to the transgene sequence or positioning may be necessary to achieve desired vector characteristics (Figure 2). Systematic comparison of different positions for checkpoint antibody inserts showed optimal results for the H-ATU (unpublished data). As BTE inserts have comparable properties (size and immunoglobulin domains), MV-BTE vectors were cloned analogously38.
Rescue of infectious particles from virus cDNA (step 2.1) may require several attempts for some constructs. Adjusting cell numbers can optimize cell density for syncytia formation. While a certain density is necessary for efficient cell-to-cell spread and fusion of cell membranes, contact inhibition reduces viral replication. Further, the number of infectious particles may not be sufficient to induce visible cell fusion. However, as viruses can be present in absence of syncytia, rescue samples can nevertheless be harvested and transferred to fresh producer cells for potential propagation. Inefficient transfection due to poor DNA quality or unsuitable or degraded transfection reagents represent typical problems that are relatively easy to assess and remedy by generating new DNA preparations and testing different reagents, respectively.
Successful virus propagation (step 2.2) is crucially dependent on the conditions determining viral replication and cell lysis. Using low passage numbers of producer cells is recommended, and infected cells need to be regularly checked for syncytia progression. Adjusting cell numbers, temperatures, and incubation times may be required to balance viral spread vs. cell proliferation.
A crucial limitation of the described method of virus production is the preparation of virus suspensions from crude cell lysates. Researchers should be aware of the fact that the virus suspension contains cellular factors. Virus particles can be concentrated via density gradient/sucrose cushion ultracentrifugation at the expense of total number of infectious particles and also potentially increasing concentrations of cellular remnants. Virus can also be concentrated from infected cell supernatants, increasing purity but reducing overall yield. GMP and large scale production of paramyxoviruses is typically performed using multiple layers of producer cells seeded on a filament net in bioreactors for increased titer yield63. Accurate monitoring, continuous media exchange, serum-free conditions, and subsequent filtration steps ensure high purity of produced virus.
Evaluation of viral titers (steps 2.3 and 3.1) via the described syncytia counting method is not precise, but it is easy to perform and sufficiently accurate when using adequate numbers of technical replicates. When evaluating OV-mediated cytotoxicity (step 3.2), limitations of available assays need to be considered. Metabolic assays do not distinguish between cytostatic and cytolytic effects of experimental treatments. To measure cytolysis, an LDH release assay may be performed. However, both types of assays can be affected by contents of virus preparations from crude cell lysates (Figure 6).
Isolation of proteins expressed by virus-infected cells (step 4.1) can vary greatly in yield and purity, depending on the respective transgene and virus used as well as on technical accuracy. Thus, quality control of protein purification is crucial for the evaluation of related experimental results.
As an exhaustive description of potential functional assays covering the wide variety of possible immunomodulators is beyond the scope of this publication, this protocol focuses on ex vivo methodology to measure cellular cytotoxicity (step 4.3). Cellular cytotoxicity, especially by CD8+ T cells, is an important mediator of immunological tumor control in successful immunotherapies64. This has important implications for current immunotherapy approaches using antibodies to enhance anti-tumor immune responses in general and bispecific T cell engagers in particular. Local BTE expression by oncolytic vectors has yielded promising results in several preclinical studies, including RNA38 and DNA viruses65,66,67,68.
Due to the complex interactions of tumor, virus, transgene product, and host immune system in living organisms, validation of immunomodulator-encoding oncolytic vectors in cell culture as demonstrated here is not sufficient to predict therapeutic outcomes. Importantly, the proposed isolated assessment of vector and immunomodulator functions, respectively, is essential for proof of concept but fails to describe potential synergies and complex interactions within the tumor microenvironment. Testing for both toxicity and efficacy in relevant in vivo models is crucial for further development of novel recombinant vector constructs but is not easily accomplished. Immunological safety is regularly monitored in non-human primates such as macaques, and mouse models are used for biodistribution and efficacy analyses20,69. However, many of these models have limitations regarding the expression of virus receptors in host tissues and host permissiveness, and some only insufficiently mimic the complex interplay of OV, tumor, and the immune microenvironment. Thus, careful consideration of appropriate models is mandatory.
MV-BTE treatment has previously been evaluated in human xenograft and syngeneic mouse models. No BTE transgene products were detectable in serum of mice treated with BTE-encoding MV38, indicating successful prevention of systemic exposure even without further attenuation of the naturally oncotropic vaccine strain virus. Local expression is crucial for OV-encoded immunomodulators which are toxic when administered systemically. If necessary, tumor specificity can be enhanced by re-targeting to cell surface markers of choice70,71,72 and microRNA-based de-targeting for enhanced oncotropism73,74 (reviewed by Ruiz and Russell75). Additional modifications may be introduced to optimize vectors for specific therapeutic uses (reviewed by Miest and Cattaneo76), including insertion of transgenes for diagnostic purposes77,78 or prodrug conversion to minimize side effects of chemotherapy79,80, introduction of safety switches81, and targeting of tumor stroma or vasculature (e.g., via bispecific antibodies82).
Aside from genetic modification of the viral vector, combination regimens with chimeric antigen receptor (CAR) T cell transfer83, chemotherapy84,85,86, or radiotherapy87,88,89 further augment the repertoire of OVT. As MV immunity is highly prevalent, strategies to circumvent antibody neutralization have been developed, including exchanging envelope glycoproteins for those of related paramyxoviruses90, polymer coating of particles, using cell carriers to deliver viruses, and transient immunosuppression (reviewed by Russell et al.6). Further development of advanced OV immunotherapy regimens requires testing of safety and therapeutic efficacy in appropriate animal models, with patient material, and, ultimately, in controlled clinical trials.
Given the plethora of possible combinations, comprehensive testing is not feasible. Mathematical modeling can aid in prioritization of potential combination regimens as well as their respective dosing and scheduling by predicting treatment outcomes in silico (reviewed by Santiago et al.91). When testing efficacies of novel, rationally designed vectors, sound accompanying translational research is instrumental for a deeper understanding of underlying virological and immunological processes. This is crucial for the generation of appropriate models, information on further potential targets, and advancement in the field in general. In conclusion, firsthand insight into relevant methods of vector design, generation, and characterization in this line of work will accelerate development and support exploration of novel therapeutics for future clinical translation.
The authors have nothing to disclose.
These methods were established in the Virotherapy Group led by Prof. Dr. Dr. Guy Ungerechts at the National Center for Tumor Diseases in Heidelberg. We are indebted to him and all members of the laboratory team, especially Dr. Tobias Speck, Dr. Rūta Veinalde, Judith Förster, Birgit Hoyler, and Jessica Albert. This work was supported by the Else Kröner-Fresenius-Stiftung (Grant 2015_A78 to C.E. Engeland) and the German National Science Foundation (DFG, grant EN 1119/2-1 to C.E. Engeland). J.P.W. Heidbuechel receives a stipend by the Helmholtz International Graduate School for Cancer Research.
Rapid DNA Dephos & Ligation Kit | Roche Life Science, Mannheim, Germany | 4898117001 | |
CloneJET PCR Cloning Kit | Thermo Fisher Scientific, St. Leon-Rot | K1231 | |
Agarose | Sigma-Aldrich, Taufkirchen, Germany | A9539-500G | |
QIAquick Gel Extraction Kit | QIAGEN, Hilden, Germany | 28704 | |
NEB 10-beta Competent E. coli | New England Biolabs (NEB), Frankfurt/Main, Germany | C3019I | |
LB medium after Lennox | Carl Roth, Karlsruhe, Germany | X964.1 | |
Ampicillin | Carl Roth, Karlsruhe, Germany | HP62.1 | |
QIAquick Miniprep Kit | QIAGEN, Hilden, Germany | 27104 | |
Restriction enzyme HindIII-HF | New England Biolabs (NEB), Frankfurt/Main, Germany | R3104S | |
Dulbecco's Modified Eagle's Medium (DMEM) | Invitrogen, Darmstadt, Germany | 31966-021 | |
Fetal bovine serum (FBS) | Biosera, Boussens, France | FB-1280/500 | |
FugeneHD | Promega, Mannheim, Germany | E2311 | may be replaced by transfection reagent of choice |
Kanamycin | Sigma-Aldrich, Taufkirchen, Germany | K0129 | |
Vero cells | ATCC, Manassas, VA, USA | CCL81 | |
B16-CD46/ B16-CD20-CD46 | J. Heidbuechel, DKFZ Heidelberg | available upon request | |
Granta-519 | DSMZ, Braunschweig, Germany | ACC 342 | |
Opti-MEM (serum-free medium) | Gibco Life Technologies, Darmstadt, Germany | 31985070 | |
Colorimetric Cell Viability Kit III (XTT) | PromoKine, Heidelberg, Germany | PK-CA20-300-1000 | includes XTT reagent |
Dulbecco's Phosphate-Buffered Saline (PBS) | Gibco Life Technologies, Darmstadt, Germany | 14190-094 | |
QIAquick Ni-NTA Spin Columns | QIAGEN, Hilden, Germany | 31014 | |
Sodium chloride | Carl Roth, Karlsruhe, Germany | 3957.3 | |
Imidazole | Carl Roth, Karlsruhe, Germany | I5513-25G | |
Amicon Ultra-15, PLGC Ultracel-PL Membran, 10 kDa | Merck, Darmstadt, Germany | UFC901024 | |
BCA Protein Assay Kit | Merck Milipore | 71285-3 | |
IgG from human serum | Sigma-Aldrich, Taufkirchen, Germany | I4506 | |
Anti-HA-PE | Miltenyi Biotech, Bergisch Gladbach, Germany | 130-092-257 | RRID: AB_871939 |
Mouse IgG1, kappa Isotype Control, Phycoerythrin Conjugated, Clone MOPC-21 antibody | BD Biosciences, Heidelberg, Germany | 555749 | RRID: AB_396091 |
Anti-HA-biotin antibody, clone 3F10 | Sigma-Aldrich, Taufkirchen, Germany | 12158167001 | RRID: AB_390915 |
Anti-Biotin MicroBeads | Miltenyi Biotech, Bergisch Gladbach, Germany | 130-090-485 | |
MS Columns | Miltenyi Biotech, Bergisch Gladbach, Germany | 130-042-201 | |
MiniMACS Separator | Miltenyi Biotech, Bergisch Gladbach, Germany | 130-042-102 | |
MACS MultiStand | Miltenyi Biotech, Bergisch Gladbach, Germany | 130-042-303 | |
RIPA buffer | Rockland Immunochemicals, Gilbertsville, PA, USA | MB-030-0050 | |
CytoTox 96 Non-Radioactive Cytotoxicity Assay | Promega, Mannheim, Germany | G1780 | includes 10x lysis solution, substrate solution (substrate mix and assay buffer), and stop solution |
Cell lifter | Corning, Reynosa, Mexico | 3008 | |
10 cm dishes | Corning, Oneonta, NY, USA | 430167 | |
15 cm dishes | Greiner Bio-One, Frickenhausen, Germany | 639160 | |
96-well plates, U-bottom | TPP, Trasadingen, Switzerland | 92097 | |
96-well plates, flat bottom | Neolab, Heidelberg, Germany | 353072 | |
6-well plates | Neolab, Heidelberg, Germany | 353046 | |
12-well plates | Neolab, Heidelberg, Germany | 353043 | |
50 mL tubes | nerbe plus, Winsen/Luhe, Germany | 02-572-3001 | |
T175 cell culture flasks | Thermo Fisher Scientific, St. Leon-Rot | 159910 | |
0.22 µm filters | Merck, Darmstadt, Germany | SLGPM33RS |