Cancer Immunotherapy - Methods and Protocols by Velia Siciliano,Francesca Ceroni 2024

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Edited by : 

Velia Siciliano 

Synthetic and Systems Biology for Biomedicine, Isituto Italiano di Tecnologia-IIT, Napoli, Napoli, Italy

Francesca Ceroni 

Department of Chemical Engineering, Imperial College, London,    

  Pages:  313                                                                                                                                     

Contents

Preface: Cancer Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 - Isolation of Live Immune Cells from the Tumor Microenvironment by FACS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Aikaterini Kafka, Christos Ermogenous, and Luigi Ombrato 

2 -  In Vitro Evaluation of Cancer Cell Immunogenicity and Antigen-Specific T-Cell Cytotoxicity by Flow Cytometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Martina Musella, Nicoletta Manduca, Ester Maccafeo, Eliana Ruggiero, and Antonella Sistigu 

3 - Retroviral Transduction of Human Primary T Cells Followed by Real-Time T-Cell-Mediated Cancer Cell Cytolysis Analysis . . . . . . . . . . . . . . . . 29 Anne Rahbech, Reno Debets, Per thor Straten, and Marlies J. W. Peeters 

4  -Expansion and Retroviral Transduction of Primary Murine T Cells for CAR T-Cell Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Pauline Loos, Lauralie Short, Gillian Savage, and Laura Evgin 

5 - In Situ Decellularization of Tissues Applied to the Topographical Analysis of Tumor-Associated Extracellular Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Alejandro E. Mayorca-Guiliani 

6 - Monitoring Cell Cytoskeleton Variations upon Piezoelectric Stimulation: Implications for the Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 O¨ zlem S¸ en, Carlotta Pucci, and Gianni Ciofani 

7 - Preparation Method and In Vitro Characterization of Nanoparticles Sensitive to Tumor Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Naym Blal and Daniela Guarnier-  8 A New Microfluidic Device to Facilitate Functional Precision Medicine Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Albert Manzano-Mun˜oz, Jose Yeste, Marı´a A. Ortega, Josep Samitier, Javier Ram'on-Azc'on, and Joan Montero

 9 -  Kinetic Detection of Apoptosis Events Via Caspase 3/7 Activation in a Tumor-Immune Microenvironment on a Chip. . . . . . . . . . . . . . . . . . . . . . . . . . 109 Francesca Romana Bertani, Farnaz Dabbagh Moghaddam, Cristiano Panella, Sara Maria Giannitelli, Valentina Peluzzi, Annamaria Gerardino, Alberto Rainer, Giuseppe Roscilli, Adele De Ninno, and Luca Businaro 

10 -  Hypoxic 3D Tumor Model for Evaluating of CAR-T Cell Therapy In Vitro. . . . 119 Jeong Min Oh and Keyue Shen 

11 -  Rapid Screening of CAR T Cell Functional Improvement Strategies by Highly Multiplexed Single-Cell Secretomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Dragana Slavkovic-Lukic, Jessica Fioravanti, Azucena Martı´n-Santos, Edward Han, Jing Zhou, and Luca Gattinoni

12 -  Genome Editing in CAR-T Cells Using CRISPR/Cas9 Technology . . . . . . . . . . 151 Irene Andreu-Saumell, Alba Rodriguez-Garcia, and Sonia Guedan 

13 -  Genetic Modification of Tumor-Infiltrating Lymphocytes, Peripheral T Cells, and T-Cell Model Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Hadas Weinstein-Marom, Dayana Blokon-Kogan, Maya Levi-Mann, Chaja Katzman, Shira Shalev, Masha Zaitsev, Michal J. Besser, Ronnie Shapira-Frommer, Gideon Gross, Orit Itzhaki, and Lior Nissim 

14 -  Transposon-Based Manufacturing of Human CAR-T Cells . . . . . . . . . . . . . . . . . . 187 Megan Tennant and Richard O’Neil 

15  - Redirecting Human Conventional and Regulatory T Cells Using Chimeric Antigen Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Capers M. Zimmerman, Rob A. Robino, Russell W. Cochrane, Matthew D. Dominguez, and Leonardo M. R. Ferreira 

16  - How to Test Human CAR T Cells in Solid Tumors, the Next Frontier of CAR T Cell Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 Russell W. Cochrane, Andrew Fiorentino, Eva Allen, Rob A. Robino, Jaime Quiroga, and Leonardo M. R. Ferreira 

17  - Nano-optogenetic CAR-T Cell Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Nhung Thi Nguyen, Siyao Liu, Gang Han, Yubin Zhou, and Kai Huang 

18 -  A Nonviral piggyBac Transposon-Mediated Method to Generate Large-Scale CAR-NK Cells from Human Peripheral Blood Primary NK Cells . . . . . . . . . . . . . 279 Zhicheng Du, Tianzhi Zhao, Xianjin Chen, Shijun Zha, and Shu Wang 

19 -  Engineering Probiotic E. coli Nissle 1917 for Release of Therapeutic Nanobodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 Candice Gurbatri and Tal Danino Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307

Preface

Over the past years, immunotherapy has emerged as a ground-breaking technology for cancer treatment, in the attempt of removing the breaks that hold the full potential of immune cells activity for targeted tumor cell killing. A variety of approaches have been developed to date, from the design of organoid models that mimic disease state to adoption of cell engineering approaches where immune cells are equipped with novel or more robust capabilities for targeted immune response. By bringing together some of the most prominent scientists in the field, this book aims at providing an overview of the different areas that are converging to develop the next generation of immunotherapy treatments. The book will walk the readers across state-ofthe-art methods for the analysis and characterization of the interactions between tumor and immune cells, and cell engineering tools for cancer treatment, to provide a unique and compelling set of techniques instrumental to work with, and engineer, immune cells.

 Challenges are still standing in the effort for a clear understanding of the interactions occurring among the different players within the tumor microenvironment, critical to develop improved therapies. Chapters 1 and 2 introduce flow cytometry-based assays to isolate immune cells from the tumor microenvironment or to evaluate cancer cell immunogenicity of ovalbumin (OVA)-specific CD8 OT-1 T cells exposed to OVA-expressing MCA205 sarcoma cells. In Chap. 3, a T-cell receptor specific for melanoma antigen gp100 is presented and its killing capabilities characterized by the xCELLingence system for real time cell analysis. Chapter 4 describes a protocol for the generation and expansion of CAR T cells using primary mouse T cells. Novel methodologies that support better understanding and characterization of the features of the tumor environment and the interaction of the different cellular types within it are awaited. Chapters 5, 6 and 7 focus on this, with Chap. 5 introducing in situ decellularization of tissues as a mean to enable the mapping of the extra cellular matrix that is key in supporting tumor growth in vivo, while in Chap. 6, piezoelectric stimulation is described as a novel approach to study the migration and invasion ability of tumor cells. Chapter 7 describes protocols to characterize the physical-chemical properties and therapeutic potential of nanoparticles in vitro on three-dimensional (3D) tumor spheroids.

 Microfluidics has witnessed tremendous development over the last decade and application across different fields. In immunotherapy, microfluidics can be adopted for the live analysis of cell-to-cell interactions but also for miniaturization of experiments decreasing the need for starting material, often difficult to isolate. This is the focus of Chap. 8 where a novel microfluidic platform is presented that exposes cancer cells to a linear increasing concentration of a given cytotoxic agent, allowing its assessment using fluorescent probes. The feature of this microfluidic design, requiring minimal handling and just a basic equipment, could facilitate the implementation of functional assays in hospitals to benefit cancer patients. Chapter 9 introduces kinetic detection of apoptosis events via caspase activation in a tumorimmune microenvironment on a chip, while Chap. 10 demonstrates how to establish a hypoxic 3-dimensional (3-D) tumor model using a cleanroom-free, micromilling-based microdevice and assesses the efficacy of the combinatorial treatment with CAR-T cells and PD-1/PD-L1 inhibition. v vi Preface: Cancer Immun

As it is well known, CAR-T cell therapy is revolutionizing the treatment of hematologic malignancies. However, there are still many challenges ahead before CAR-T cells can be used effectively to treat solid tumors and certain hematologic cancers, such as T-cell malignancies. Thus, several strategies are being investigated to improve the response to T cell therapies. For example, the functional fitness of CAR T cells directly correlates with their clinical efficacy. A standing limitation is the screening of approaches to improve cellular fitness in vivo, which is expensive and time-consuming. Chapter 11 describes a highly multiplexed single-cell secretomic assay based on the IsoLight platform to rapidly evaluate the impact of new pharmacologic or gene-engineering approaches aiming at improving CAR T cell function.

Further, several strategies are explored for immune cell engineering, from retroviral transduction to genome editing to knock-out or knock-in genes. Chapters 12, 13, 14, and 15 describe protocols for T cell engineering by using CRISPR-Cas9 technology, retroviruses, mRNA transfection, transposons, and lentiviruses respectively, while Chap. 16 describes procedures to evaluate human CAR T cells in solid tumors. Considering current concerns related to the control of transgene expression by engineered T cells such as cytokine release syndrome (CRS) and “on-target, off-tumor” cytotoxicity, we include in this book, Chapter 17 as an example of spatio-temporal control of T cell activity by nanooptogenetics. This system comprises synthetic light-sensitive CAR T cells and nanoparticles acting as in situ nano-transducer, allowing near-infrared light to wirelessly control CAR T cell immunotherapy.

Besides CAR T cells, engineering of cell-based therapy is further branching out to other immune cells such as natural killer cells (NK), among the first to infiltrate and fight tumors. Current methods of producing large-scale CAR-NK cells mainly rely on mRNA transfection and viral vector transduction. However, mRNA CAR-NK cells were not stable in CAR expression while viral vector transduction mostly ended up with low efficiency. Chapter 18 describes an optimized protocol to generate CAR-NK cells by using the piggyBac transposon system via electroporation and to expand the engineered CAR-NK cells in large-scale together with artificial antigen-presenting feeder cells. This method can stably engineer human primary NK cells with high efficiency and supply sufficient scale of engineered CAR-NK cells for the future possible clinical applications.

 Finally, we wanted to conclude with an example of what the integration of synthetic biology and immunology is further leading to. In Chap. 19, a novel approach for immunotherapy is presented based on bioengineered probiotics. Since probiotics have tumorcolonizing capabilities, a new probiotic E. coli Nissle 1917-based platform was developed, encoding a synchronized lysis mechanism for the localized and sustained release of blocking nanobodies against immune checkpoint molecules like programmed cell death proteinligand 1 and cytotoxic T lymphocyte associated protein-4. We believe that this comprehensive and detailed protocols will benefit bioengineers approaching to immunology and vice versa, creating a blossoming interdisciplinary community of synthetic immunologists.