Name: paramyxoviruses for tumor-targeted immunomodulation: design and evaluation ex vivo
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo at JoVE.com

These methods were established in the Virotherapy Group led by Prof. Dr.&#160;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. R&#363;ta Veinalde, Judith F&#246;rster, Birgit Hoyler, and Jessica Albert. This work was supported by the Else Kr&#246;ner-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.

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
<ol>
	<li>[O] Design insert sequence.
	<ol>
		<li>[O] Decide on an immunomodulator of interest based on literature research or on exploratory data such as genetic screens41 and derive ...

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 advantag...

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&#160;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&#160;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&#160;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).

<table border="0" cellpadding="0" cellspacing="0" style="width:833px;" width="833">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Rapid DNA Dephos &#38; Ligation Kit</td>
			<td style="width:312px;">Roche Life Science, Mannheim, Germany</td>
			<td style="width:123px;">4898117001</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">CloneJET PCR Cloning Kit</td>
			<td style="width:312px;">Thermo Fisher Scientific, St. Leon-Rot</td>
			<td style="width:123px;">K1231</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Agarose</td>
			<td style="width:312px;">Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:123px;">A9539-500G</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">QIAquick Gel Extraction Kit</td>
			<td style="width:312px;">QIAGEN, Hilden, Germany</td>
			<td style="width:123px;">28704</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">NEB 10-beta Competent E. coli</td>
			<td style="width:312px;">New England Biolabs (NEB), Frankfurt/Main, Germany</td>
			<td style="width:123px;">C3019I</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">LB medium after Lennox</td>
			<td style="width:312px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:123px;">X964.1</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Ampicillin</td>
			<td style="width:312px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:123px;">HP62.1</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">QIAquick Miniprep Kit</td>
			<td style="width:312px;">QIAGEN, Hilden, Germany</td>
			<td style="width:123px;">27104</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Restriction enzyme HindIII-HF</td>
			<td style="width:312px;">New England Biolabs (NEB), Frankfurt/Main, Germany</td>
			<td style="width:123px;">R3104S</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td style="width:312px;">Invitrogen, Darmstadt, Germany</td>
			<td style="width:123px;">31966-021</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Fetal bovine serum (FBS)</td>
			<td style="width:312px;">Biosera, Boussens, France</td>
			<td style="width:123px;">FB-1280/500</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:244px;">FugeneHD</td>
			<td style="width:312px;">Promega, Mannheim, Germany</td>
			<td style="width:123px;">E2311</td>
			<td style="width:155px;">may be replaced by transfection reagent of choice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Kanamycin</td>
			<td style="width:312px;">Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:123px;">K0129</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Vero cells</td>
			<td style="width:312px;">ATCC, Manassas, VA, USA</td>
			<td style="width:123px;">CCL81</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">B16-CD46/ B16-CD20-CD46</td>
			<td style="width:312px;">J. Heidbuechel, DKFZ Heidelberg</td>
			<td style="width:123px;"></td>
			<td style="width:155px;">available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Granta-519</td>
			<td style="width:312px;">DSMZ, Braunschweig, Germany</td>
			<td style="width:123px;">ACC 342</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Opti-MEM (serum-free medium)</td>
			<td style="width:312px;">Gibco Life Technologies, Darmstadt, Germany</td>
			<td style="width:123px;">31985070</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Colorimetric Cell Viability Kit III (XTT)</td>
			<td style="width:312px;">PromoKine, Heidelberg, Germany</td>
			<td style="width:123px;">PK-CA20-300-1000</td>
			<td style="width:155px;">includes XTT reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Dulbecco&#39;s Phosphate-Buffered Saline (PBS)</td>
			<td style="width:312px;">Gibco Life Technologies, Darmstadt, Germany</td>
			<td style="width:123px;">14190-094</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">QIAquick Ni-NTA Spin Columns</td>
			<td style="width:312px;">QIAGEN, Hilden, Germany</td>
			<td style="width:123px;">31014</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Sodium chloride</td>
			<td style="width:312px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:123px;">3957.3</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Imidazole</td>
			<td style="width:312px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:123px;">I5513-25G</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Amicon Ultra-15, PLGC Ultracel-PL Membran, 10&#160;kDa</td>
			<td style="width:312px;">Merck, Darmstadt, Germany</td>
			<td style="width:123px;">UFC901024</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">BCA Protein Assay Kit</td>
			<td style="width:312px;">Merck Milipore</td>
			<td style="width:123px;">71285-3</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">IgG from human serum</td>
			<td style="width:312px;">Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:123px;">I4506</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Anti-HA-PE</td>
			<td style="width:312px;">Miltenyi Biotech, Bergisch Gladbach, Germany</td>
			<td style="width:123px;">130-092-257</td>
			<td style="width:155px;">RRID: AB_871939</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:244px;">Mouse IgG1, kappa Isotype Control, Phycoerythrin Conjugated, Clone MOPC-21 antibody</td>
			<td style="width:312px;">BD Biosciences, Heidelberg, Germany</td>
			<td style="width:123px;">555749</td>
			<td style="width:155px;">RRID: AB_396091</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Anti-HA-biotin antibody, clone 3F10</td>
			<td style="width:312px;">Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:123px;">12158167001</td>
			<td style="width:155px;">RRID: AB_390915</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Anti-Biotin MicroBeads</td>
			<td style="width:312px;">Miltenyi Biotech, Bergisch Gladbach, Germany</td>
			<td style="width:123px;">130-090-485</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">MS Columns</td>
			<td style="width:312px;">Miltenyi Biotech, Bergisch Gladbach, Germany</td>
			<td style="width:123px;">130-042-201</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">MiniMACS Separator</td>
			<td style="width:312px;">Miltenyi Biotech, Bergisch Gladbach, Germany</td>
			<td style="width:123px;">130-042-102</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">MACS MultiStand</td>
			<td style="width:312px;">Miltenyi Biotech, Bergisch Gladbach, Germany</td>
			<td style="width:123px;">130-042-303</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">RIPA buffer</td>
			<td style="width:312px;">Rockland Immunochemicals, Gilbertsville, PA, USA</td>
			<td style="width:123px;">MB-030-0050</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:244px;">CytoTox 96 Non-Radioactive Cytotoxicity Assay</td>
			<td style="width:312px;">Promega, Mannheim, Germany</td>
			<td style="width:123px;">G1780</td>
			<td style="width:155px;">includes 10x lysis solution, substrate solution (substrate mix and assay buffer), and stop solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Cell lifter</td>
			<td style="width:312px;">Corning, Reynosa, Mexico</td>
			<td style="width:123px;">3008</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">10 cm dishes</td>
			<td style="width:312px;">Corning, Oneonta, NY, USA</td>
			<td style="width:123px;">430167</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">15 cm dishes</td>
			<td style="width:312px;">Greiner Bio-One, Frickenhausen, Germany</td>
			<td style="width:123px;">639160</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">96-well plates, U-bottom</td>
			<td style="width:312px;">TPP, Trasadingen, Switzerland</td>
			<td style="width:123px;">92097</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">96-well plates, flat bottom</td>
			<td style="width:312px;">Neolab, Heidelberg, Germany</td>
			<td style="width:123px;">353072</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">6-well plates</td>
			<td style="width:312px;">Neolab, Heidelberg, Germany</td>
			<td style="width:123px;">353046</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">12-well plates</td>
			<td style="width:312px;">Neolab, Heidelberg, Germany</td>
			<td style="width:123px;">353043</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">50 mL tubes</td>
			<td style="width:312px;">nerbe plus, Winsen/Luhe, Germany</td>
			<td style="width:123px;">02-572-3001</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">T175 cell culture flasks</td>
			<td style="width:312px;">Thermo Fisher Scientific, St. Leon-Rot</td>
			<td style="width:123px;">159910</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">0.22 &#181;m filters</td>
			<td style="width:312px;">Merck, Darmstadt, Germany</td>
			<td style="width:123px;">SLGPM33RS</td>
			<td style="width:155px;"></td>
		</tr>
	</tbody>
</table>

paramyxoviruses for tumor-targeted immunomodulation: design and evaluation ex vivo

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.

Figure 1&#160;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 fe...

Watch this Scientific Journal Video about Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo at JoVE.com

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

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 ...

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