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

Summary

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.

Abstract

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.

Introduction

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 stimulation^1,2,3,4. While many early OV studies used unm....

Protocol

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

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

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
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