NK Cells in Cancer Immunotherapy: Successes and Challenges

By Yuman Fong

NK Cells in Cancer Immunotherapy: Successes and Challenges explains the latest immunotherapeutic strategies, focusing on NK cells to allow the best and precise combination treatments to cancer patients. The book provides existing background knowledge in the field of immunotherapy and discusses future areas of research required to carry out cutting-edge, validated therapies. Chapters cover advances in immunotherapeutic strategies, in particular, the use of NK cells with and without T-cell therapy in the treatment of cancer. The book is a valuable resource for cancer researchers, oncologists, graduate students and those interested in learning more about novel strategies to treat cancer patients.

Immunotherapy is fast becoming the method of choice for cancer therapy. Although remarkable advances have been made in the field of immunotherapy, there are significant challenges and difficulties ahead since many of the current immunotherapeutic strategies do not provide long-lasting treatment strategies, and therefore are not very effective.

• Covers CAR/T and CAR/NK and adoptive NK cell therapy with and without T cell therapies
• Discusses basic biology of NK cells and mouse models of human cancers and the role of NK cells in metastatic cancer and in cancer stem cells
• Encompasses information on combination therapies using check point inhibition, adoptive transfer of cytotoxic effector cells, chemotherapeutic drugs and activating and inhibitory antibodies

• Language: English
• Publisher: Academic Press
• Release date: Dec 6, 2022
• ISBN: 9780128226261

Book preview

9780128226261_FC

NK Cells in Cancer Immunotherapy: Successes and Challenges

First Edition

Anahid Jewett, Ph.D., M.P.H.

Division of Oral Biology and Medicine, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, University of California Los Angeles (UCLA), Los Angeles, CA, United States

The Jonsson Comprehensive Cancer Center, UCLA School of Dentistry and Medicine, Los Angeles, CA, United States

Yuman Fong, M.D.

Department of Surgery, City of Hope Comprehensive Cancer Center, Duarte, CA, United States

Table of Contents

Cover

Title page

Copyright

Cover Image Insert

Hypothetical role of NK cells in differentiation of tumor cells, increased susceptibility of tumor cells to chemotherapy or radiation and lower risk of metastasis

Aims and Scope of Series Breaking Tolerance to Anti-Cancer Cell-Mediated Immunotherapy

About the Series Editor

Aims and Scope of Volume

About the Volume Editors

Preface—Cellular immunotherapies: Evolution from laboratory studies to effective human therapies

Contributors

Section I: Basics of cellular immunotherapy: Differing roles of NK and T cells in targeting cancer and their intimate synergistic interaction underscores the requirement for the use of both cell types in successful cancer treatment

Chapter 1: Multifaceted nature of natural killer cells: Potential mode of interaction and shaping of stem cells

Abstract

Acknowledgments

Conflict of interest

Natural killer cells: Overview and background

Two faces of NK cells: Concept of split anergy in NK cells and its potential role in tumor differentiation

The rationale for the functional activation of NK cells in many gene knockout mice: Potential common mechanism of activation

Dysfunctional NK cells in cancer patients: Defects in NK cells to lyse and differentiate CSCs

Suppression of antitumor immune function and change in NK cell phenotype in tumor microenvironment: Could compromised NK function lead to dysregulated immune function in tumor microenvironment?

Tumor-associated stromal cells may shape the function of NK cells

Function of NK cells in tumor-bearing humanized-BLT mice mirrors those of the cancer patients

Novel strategy to expand supercharged NK cells for immunotherapy using osteoclasts as feeder cells: Different efficacy of supercharged NK cell expansion and function using allogeneic vs autologous NK cells from healthy or cancer patients

Supercharged NK cells differ from primary NK cells in phenotype and function

Supercharged NK cells preferentially and rapidly expand CD8 + T cells

Functional differences of NK cells in different NK expansion platforms against CSCs/poorly differentiated tumor cells: Comparison with supercharged NK cells

Immunotherapy is essential in combination with chemotherapy: Chemotherapy targets differentiated tumors more than cancer stem-like cells

Combination of NK cell and antibody therapy: NK cells can target CSCs and their differentiated counterparts through direct lysis and/or ADCC, respectively

NK cells and the oncolytic viruses

Combination therapy with NK cells and immune checkpoint inhibitors

Conclusions

References

Chapter 2: Reversing the NK inhibitory tumor microenvironment by targeting suppressive immune effectors

Abstract

Conflict of interest

Introduction

Dysregulation of NK cell immune responses by the tumor microenvironment

Suppressive immune effectors of the tumor microenvironment

Techniques to evaluate the tumor microenvironment

Advancements in therapeutic modalities to overcome TME suppression

Perspectives and conclusions

References

Chapter 3: Natural killer cells as immunotherapeutic effectors for solid tumors

Abstract

Conflict of interest

Introduction

Sources of NK cells for immunotherapy (Table 1)

Challenges to NK cell immunotherapy in solid tumors (Table 2)

Genetic engineering of NK cells

Conclusions

References

Chapter 4: Targeting NKG2D/NKG2D ligand axis for cancer immunotherapy

Abstract

Conflict of interest

Introduction

NKG2D

NKG2D in antitumor immunity

Conclusions

References

Chapter 5: Chimeric antigen receptor-modified cells for the treatment of solid tumors: First steps in a thousand-mile march

Abstract

Acknowledgments

Conflict of interest

Introduction

CAR T-cell therapy experience in solid tumors

Overcoming challenges facing CAR therapy in solid tumors

Beyond CAR T-cell therapy

Conclusion

References

Chapter 6: Tumor-infiltrating lymphocyte (TIL) therapy

Abstract

Acknowledgments

Conflict of interest

Introduction

Improving antitumor efficacy of TIL ACT by targeting neoantigens

Impact of cellular phenotype upon TIL ACT longevity

Improvement in TIL manufacturing and modern-day clinical trials

TILs and solid tumors other than melanoma

Summary

References

Chapter 7: Biology and status of chimeric antigen receptor-engineered T cell therapy

Abstract

Conflict of interest

Introduction

Generations of CAR T cells

FDA approved CAR T cells in hematologic malignancies

Challenges in solid tumor targeting with CAR T cell therapy

Physical and metabolic barriers in the solid tumor microenvironment

The immunosuppressive TME

Solid tumor antigen heterogeneity and antigen escape

Future perspective of CAR T cell therapy approaches to tackle solid tumors

References

Section II: Process and trials optimization: Diagnostics, readouts, route, and production to optimize cell therapy

Chapter 8: Optimization of production for cell therapies

Abstract

Conflict of interest

Keep the end goal in mind from early stage: Define the target product profile

Process design: Take a quality by design approach

Get to the goal: Many decisions to make

Understand the analytical needs

Identify and overcome the distribution, thawing, and dispensing challenges

Conclusion

References

Chapter 9: Lymphodepletion and cellular immunotherapy

Abstract

Conflict of interest

Introduction

Solid tumors

Conclusion

References

Chapter 10: Imaging the immune cell in immunotherapy

Abstract

Acknowledgments

Conflict of interest

Imaging objectives from an immunological perspective

Imaging modalities and labeling strategies for cancer immunotherapy

Application of the Imaging Toolbox toward cancer immunotherapy

What the future holds

References

Chapter 11: Radiologic assessment of tumor response to immunotherapy and its complications

Abstract

Acknowledgments

Conflict of interest

Introduction

Uses of medical imaging in immunotherapy

Challenges to medical imaging presented by immunotherapy

Methods of measurement of tumor response

Future directions for imaging tumor response to immunotherapy

Conclusions

References

Chapter 12: Novel cell delivery systems: Intracranial and intrathecal

Abstract

Conflict of interest

Tumors of the central nervous system

Blood brain barrier (BBB)

Direct delivery

Methods to disrupt the BBB

Conclusion

References

Chapter 13: Diagnostic methods to assess the numbers, phenotype, and function of primary and engineered NK cells: Methods to predict prognosis and treatment outcome

Abstract

Conflict of interest

Introduction

Major parameters to assess NK cell functions

Methodologies to assess NK cell surface markers, cytotoxicity, and secretion

Significance of NK cell diagnostics in the field of cancer

NK cell diagnostics: Future of cancer diagnostics

References

Section III: Patient trials and combinational strategies in cellular immunotherapy; Successful cell therapy may depend on the selection of treatment strategies synergistic with cell therapy

Chapter 14: Combination of NK cell immunotherapy with chemotherapy and radiation enhances NK cell therapy and provides improved prognosis in cancer patients and in humanized BLT mouse model system

Abstract

Acknowledgments

Author contributions

Statement of originality

Conflict of interest statement

Introduction

Preclinical and clinical development of NK cell-based immunotherapies

Rationale for NK cell-based immunotherapy

Rationale for combination of chemotherapy with NK-based therapy

Combination of cisplatin with supercharged NK cell immunotherapy enhances NK cell mediated killing and increases the secretion of IFN-γ in humanized-BLT mice

Rationale for combination of targeted therapy with NK-based therapy

Rationale for combination of radiation therapy and NK cell therapy

Conclusion

References

Chapter 15: Combining oncolytic viruses with immune cell therapy as treatments for cancer: OV, CAR T-cell, and NK combinations

Abstract

Conflict of interest

Introduction

CAR-T cell therapy and oncolytic viruses in cancer therapy

NK cells and oncolytic viruses in cancer therapy

Immune cells as carriers of OV in cancer therapy

Conclusion

References

Chapter 16: Natural killer cells in the treatment of glioblastoma: Diverse antitumor functions and potential clinical applications

Abstract

Acknowledgments

Author contributions

Conflict of interest

Introduction

Natural killer cells: Basic biology and dysfunction in cancer

Use of natural killer cell-based therapeutic platforms for cancer immunotherapy

Glioblastoma remains an incurable primary brain tumor

Natural killer cell-based immunotherapy for primary brain tumors

Preclinical tumor models and the translation of natural killer cell therapy into the clinic

Conclusions and future directions

References

Chapter 17: Immunotherapy using CAR T: What we have learned from trials and where we are heading

Abstract

Conflicts of interest

Funding support

Disclosures

Landscape of CAR T-cell clinical trials

New developments in CAR design

CAR T-cell trials vs ICI trials

Lessons learned in optimizing the clinical flow of CAR T-cell trials

Development of institutional infrastructure to conduct successful CAR T-cell trials

I. Standardized workflows across the institution that are flexible to different disease models and CAR constructs

II. Standardized grading and treatment algorithms for the management of CRS and neurotoxicity

III. Interdisciplinary collaboration

IV. Research and ancillary support

V. Ongoing education

Where we are heading

References

Chapter 18: NKT cell: Success and promises in transplantation and immunotherapy

Abstract

Acknowledgments

Conflict of interest

Biology of NKT cells

Mechanisms and relevant studies of iNKT cells suppressing GvHD in allogeneic transplantation

Mechanisms of iNKT cells in antitumor immunity

Regulation of antitumor effector cells

iNKT cell-based immunotherapy for treating cancer

Challenges and perspectives

References

Chapter 19: Tumor-infiltrating lymphocytes: Prognostic considerations and current trials as adoptive cell therapy

Abstract

Conflict of interest

Introduction

History of ACT

TILs as a prognostic factor for survival in selected cancers

Preparation of TIL for ACT

ACT therapy

Safety and tolerability of ACT

Future directions

References

Chapter 20: Molecular remission using personalized low-dose immunotherapy with minimal toxicities for poor prognosis hematological and solid tumor cancers

Abstract

Conflict of interest

Introduction

Case reports

Discussion

Conclusions

Future perspectives

References

Index

Copyright

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ISBN: 978-0-12-822620-9

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Hypothetical role of NK cells in differentiation of tumor cells, increased susceptibility of tumor cells to chemotherapy or radiation and lower risk of metastasis

Three potential treatment outcomes can be envisioned depending on the functional capacity of NK cells in patients. Since targets of NK cells are cancer stem cells/poorly differentiated tumors, when competent NK cells, having increased ability to kill and secrete IFN-g and TNF-a (A), encounter cancer stem cells/poorly differentiated tumors they will kill a large number of these tumors and differentiate the remaining tumor cells by the secretion of IFN-g and TNF-a, resulting in the generation of well-differentiated tumors. Such NK-differentiated tumors are then highly susceptible to chemotherapy or radiation and therefore are eliminated in cancer patients by the use of chemotherapy or radiation, thereby exhibiting lower risk of metastasis. Patients who may maintain high NK cytotoxicity but have low or decreased ability to secrete IFN-g and TNF-a (B) will be able to kill tumors but will have deficiency in differentiating tumors resulting in a moderately differentiated tumors. These tumors may have moderate susceptibility to chemotherapy or radiation and will pose a moderate risk for metastasis. Patients who have very low levels of cytotoxicity and secretion of IFN-g and TNF-a (C) will not be able to kill or differentiate tumors and therefore such tumors will be resistant to chemotherapy or radiation and pose a strong risk for metastasis.

fm01-9780128226209

Aims and Scope of Series Breaking Tolerance to Anti-Cancer Cell-Mediated Immunotherapy

The role of the immune system in the eradication of cancers has been investigated for several decades with controversial findings. The controversy was the result of a poor understanding of the underlying mechanisms that govern responsiveness and unresponsiveness. Hence, significant advances have been made with respect to the regulation of the host immune response against cancer and several immunotherapeutics have been recently introduced and used clinically. These include both antibody- and cell-mediated immunity targeting the cancer cells. Such immunotherapies led to significant clinical responses in various cancer types that were unresponsive to conventional therapies. However, only a subset of cancer patients responds to such immunotherapeutics and also there is a responding subset that develops resistance to further treatment. Various studies have examined potential underlying mechanisms involved in resistance and identified a variety of gene products that play pivotal roles in maintaining the resistant phenotype of the cancer cells to cell-mediated immunotherapy.

The main objective of the proposed series Breaking Tolerance to Anti-Cancer Cell-Mediated Immunotherapy is the development of individual volumes that are focused on the application of particular sensitizing agents that, when used in combination with cell-mediated immunotherapy, result in the reversal of resistance.

A variety of different classes of immunosensitizing agents has been reported. Each individual volume will focus on one class of immunosensitizing agents and their effects on the reversal of cell-mediated immune resistance in different cancers. Emphasis will be on biochemical, molecular, and genetic mechanisms by which the sensitizing agents mediate their effects individually and/or in combination with immunotherapy. Each editor will compile nonoverlapping review chapters on the therapeutic role of specific sensitizing agents used in combination with conventional immunotherapy and the reversal of resistance. There will also be an emphasis on discrimination of responses obtained in various cancer types.

The scope of the series is to provide updated information to scientists and clinicians that is valuable in their quest to gather information, carry out new investigations, and develop novel immunosensitizing agents that are both more potent and might also be active in contrast to existing ones that were not active.

Benjamin Bonavida, PhD (Series Editor)

About the Series Editor

fm01-9780128226209

Dr. Benjamin Bonavida, PhD (Series Editor), is currently distinguished research professor at the University of California, Los Angeles (UCLA). He is affiliated with the Department of Microbiology, Immunology and Molecular Genetics, UCLA David Geffen School of Medicine. His research career, thus far, has focused on investigations in the fields of basic immunochemistry and cancer immunobiology. His research investigations have ranged from the biochemical, molecular, and genetic mechanisms of cell-mediated killing and tumor cell resistance to chemo-immuno cytotoxic drugs. The reversal of tumor cell resistance was investigated by the use of various selected sensitizing agents based on molecular mechanisms of resistance. In these investigations, there was the newly characterized dysregulated NF-κB/Snail/YY1/RKIP/PTEN loop in many cancers that was reported to regulate cell survival, proliferation, invasion, metastasis, and resistance. Emphasis was focused on the roles of the tumor suppressor Raf kinase inhibitor protein (RKIP), the tumor promoter Yin Yang 1 (YY1) and the role of nitric oxide as a chemo-immuno-sensitizing factor. Many of the aforementioned studies are centered on the clinical challenging features of cancer patients’ failure to respond to both conventional and targeted therapies.

The editor has been active in the organization of regular sequential international miniconferences that are highly focused on the roles of YY1, RKIP, and nitric oxide in cancer and their potential therapeutic applications. Several books edited or coedited by the editor have been published. In addition, the editor has been the series editor of books (over 23) published by Springer on Resistance to Anti-Cancer Targeted Therapeutics. In addition, the editor is presently the series editor of three series published by Elsevier/Academic Press on Cancer Sensitizing Agents for Chemotherapy, Breaking Tolerance to Anti-Cancer Cell-Mediated Immunotherapy, and Breaking Tolerance to Antibody-Mediated Immunotherapy. Lastly, the editor is the editor-in-chief of the journal Critical Reviews in Oncogenesis. The editor has published over 500 research publications and reviews in various scientific journals of high impact.

Acknowledgments: The editor wishes to acknowledge the excellent editorial assistance of Ms. Inesa Navasardyan, who has worked diligently for the completion of this volume, by editing and formatting the various contributions to this volume. Ms. Navasardyan has recently graduated from UCLA (June 2020) and has also contributed a review chapter in this volume on triple-negative breast cancer.

The editor acknowledges the Department of Microbiology, Immunology and Molecular Genetics and the UCLA David Geffen School of Medicine for their continuous support. The editor also acknowledges the assistance of Mr. Rafael Teixeira, Acquisitions Editor for Elsevier/Academic Press, and the excellent assistance of Ms. Samantha Allard, Editorial Project Manager for Elsevier/Academic Press, for their continuous cooperation throughout the development of this book.

Aims and Scope of Volume

This year, nearly 20 million new cases of cancer will occur and 10 million people will die worldwide. Although the treatment strategies have steadily been advancing in cancer, we still lack comprehensive and durable treatment modalities to successfully prevent, treat, or cure cancer. In the past 5 years, we have witnessed approvals of four cellular therapies for clinical use, such as chimeric antigen receptor T cell (CAR-T), natural killer cell (NK), tumor-infiltrating lymphocyte (TIL), and NK-CAR therapies. We are also expecting additional cellular therapies in the coming years, and cellular immunotherapy is no longer a laboratory science but an important clinical discipline.

Despite the advancements in cancer therapy, we still have challenges to overcome since many of the current immunotherapeutic strategies are only effective in certain cancers, do not provide long-lasting remissions, and are effective in only a fraction of the cancer patients. Not only are the costs of such treatments largely prohibitive, they are also only available to certain subsets of cancer patients, and in some individuals they inflict significant collateral damage that compromises the patients’ quality of life. What we have learned from many years of investigations focusing on the in vivo immune system in cancer is a that successful treatment may require the supplementation of different immune effectors such as NK cells, CD8 + T cells, TILs, NKT, CAR-T, or CAR-NK cells to work in synergy to correct or overcome the defective nature of patients’ immune cells.

The aim of this book is to summarize and highlight the state of the field of cellular immunotherapy in cancer. The different chapters in the book have been authored by authoritative members in this field who are engaged in preclinical investigations, clinical trials, and key scientific studies. This book is divided into three sections. Section A summarizes the basics of cellular immunotherapies, delineating the basic immunobiology of NK cells and T cells in targeting cancer. In Section B of the book, the authors present processes for trial optimization including the challenges and promises of efficient production, in vivo tracking of cellular therapies, and novel diagnostics and biologic readouts. Section C is dedicated to reviewing trials and combinational strategies in cellular immunotherapy. Many of the chapters are focused on introducing cellular therapies with standard therapeutic modalities in order to chart a way forward for more effective, less toxic, and less costly cell therapies.

This book is intended to present the successes and the challenges of cellular immunotherapies for current and future translational researchers at all levels. It is meant to be a guide for graduate students and postdoctoral fellows entering the field. It is intended to summarize the current state-of-the art for the investigators and trialists active in the development and research of cellular therapies for cancer. Most of all, we intend to present this book as one of the first manuals for the emerging field of clinical cellular therapies for cancer. We hope that our audience of scientists, oncologists, radiologists, and colleagues in industry will benefit greatly from this book in not only understanding the state of technology but also effectively using the information provided in this book to advance their preclinical and clinical outcomes.

Dr. Anahid Jewett

Dr. Yuman Fong

About the Volume Editors

fm01-9780128226209

Dr. Anahid Jewett is Professor and Director of the Tumor Immunology Laboratory in the Division of Oral Biology and Medicine and Weintraub Center for Reconstructive Biotechnology at the UCLA School of Dentistry and Medicine. She has membership in the Jonsson Comprehensive Cancer Center (JCCC) and is a member of the UCLA Tumor Immunology subgroup. Her extensive educational and training background in the fields of pathology, microbiology, and immunology as well as infectious disease epidemiology positioned her well to resolve the future scientific challenges of infectious diseases as well as diseases based on inflammation, autoimmunity, and cancer in which she has a significant track record of productive publications. She is well known nationally and internationally for her contributions to the fields of NK cell biology, tumor immunology, and cancer immunotherapy. She has received a large number of honors and awards and holds memberships in many professional organizations and societies. She has chaired important committees at UCLA and elsewhere and has been instrumental in shaping the graduate studies for the health professionals at UCLA. She chairs and lectures in several graduate-level courses, and her laboratory is sought out by many foreign and domestic scholars who spend several years to receive training in her laboratory. She serves on the editorial boards of many prestigious journals and has been a reviewer on the board of the National Institute of Health and many other national and international cancer institutes, foundations, and charities. She holds many patents, has given more than 300 invited lectures and presentations nationally and internationally, and has published more than 150 high-impact journal articles, reviews, commentaries, and book chapters in the field of cancer. She has research collaborations with investigators from China, Israel, Slovenia, Mexico, Poland, Germany, Thailand, Japan, Portugal, South Korea, Austria, and Sweden to name a few. She has organized a number of conferences on cancer immunity nationally and internationally. She has trained more than 200 graduate students and health professionals in her laboratory, many of whom are leaders in their respective institutions. She has served on review panels for grants from many countries including England, France, the Netherlands, Qatar, Poland, and Israel to name a few. She has received grants from NIH as well as other foundations and institutes. She serves on the scientific boards of many companies and is a consultant to a number of investment and venture capital firms. She has served as editor-in-chief, associate editor, or guest editor for a number of journals, and has edited several journal sections for a number of distinct publishing companies. Her public outreach and education includes many interviews with celebrities, organizations, and societies in the field of cancer and infectious diseases.

One of Dr. Jewett’s major contributions to science and NK cell biology was the identification, characterization, and the establishment of the concept of split anergy in NK cells. Equally important was Dr. Jewett’s discovery demonstrating that NK cells were important for the elimination, selection, and differentiation of cancer stem cells as well as healthy stem cells. Most recently, she has identified, characterized, and patented a novel technology to expand large numbers of super-charged NK cells, which has been licensed and is on track to be used in clinical trials of cancer patients. In addition, she has developed disease-specific formulations of probiotic bacteria to restore the damaged microbiome and prevent and treat inflammation, auto-immunity, and cancer. Her novel approach to cell therapy uses a number of key therapeutic strategies used in combination to eliminate all different clones of cancer in an attempt to cure rather than short-term treatment of cancer.

Unlabelled Image

Dr. Yuman Fong is the Sangiacomo Chair and Chairman of the Department of Surgery at the City of Hope Medical Center.

Dr. Fong’s clinical expertise is in oncology and in the field of liver and pancreatic surgery. He helped usher in robotic techniques for HPB surgery. He is editor of the SAGES Atlas of Robotic Surgery. For his clinical work, he was awarded the Layton F. Rikkers Master Clinician Award from the SSAT. He has assisted in the design and deployment of many novel surgical tools. For his contributions to surgical research, he has been awarded the Stanley Dudrick Award from the American Society of Parenteral and Enteral Nutrition, The Shipley Award from the Southern Surgical Association, and the Flance-Karl Award from the American Surgical Association. His work in medical engineering has led to his election to the American Institute of Medical and Biologic Engineering.

His research has long been in the field of immunology. He was part of the team that discovered the biologic effects of tumor necrosis factor that helped establish the field of innate immunity. His current laboratory focus is on the design of gene and immune cell therapies for cancer. His leadership at the national level has included serving as the Chair of the Recombinant DNA Advisory Committee (RAC) of the National Institutes of Health. He is Founding Editor-in-Chief of Molecular Therapy Oncolytics (Cell Press, official journal of the ASGCT). He has coauthored over 1000 papers (h-index = 143. Citations 85,000) and 22 textbooks. He has also been elected to the American Society of Clinical Investigation and to the National Academy of Medicine.

In his spare time, he enjoys music, sandboarding, and ballroom dancing.

Preface—Cellular immunotherapies: Evolution from laboratory studies to effective human therapies

Cellular immunology is central to all facets of human health and pathology including cancer, infection, autoimmune disease, and recovery after trauma. Our understanding of the basic mechanisms of cellular immunity has been growing exponentially over the last half century. In oncology, until recently, studies of cellular immunity and immunotherapy have been restricted to preclinical studies and clinical trials. The last 5 years have witnessed FDA approvals of four cellular therapies for clinical use: tisagenlecleucel (Kymriah; Novartis), axicabtagene ciloleucel (Yescarta; Kite Pharma, Gilead Sciences Inc), lisocabtagene maraleucel (Breyanzi; Juno Therapeutics), and brexucabtagene autoleucel. These are all chimeric antigen receptor T cell (CAR-T) therapies and have been approved to treat a number of hematologic malignancies, including diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, and B-cell acute lymphoblastic leukemia (ALL). These new human medicines validate the field of cellular immune-therapeutics as a clinical field and hold forth the promise that many other cellular therapies [NK, NK-CAR, and tumor-infiltrating lymphocytes (TILs)] may soon become human therapies for both hematologic and solid tumors.

Cancer is in part a disease of the immune system. An effective and balanced functioning immune system should be able to recognize and remove the cells that have phenotypic changes due to gene deletions or mutations. However, when the function of immune cells, in particular NK cells and CD8 + T cells, becomes compromised, an opportunity arises for the unwanted mutated cells to survive and give rise to cancer. Although advances have been made in the field of cancer immunotherapy, we still have significant challenges to overcome since few of the current immunotherapeutic strategies provide long-lasting remissions, and all are effective in only specific cancers and a fraction of cancer patients. The high levels of adverse effects and high cost of these treatments at present are shown to result in collateral damage and significant financial constraints, respectively. The main objective of this book is to summarize the latest advancements and novel strategies for NK and T cell immunotherapies and to chart a way forward for more effective, less toxic, and less costly cell therapies.

The heterogeneous nature of cancer requires targeting by several different strategies since one strategy may be inadequate to remove all the existing clones of cancer. In this book, we introduce such combinatorial strategies. Since the discovery of NK cells, NK cell immunobiologists have been studying these cells tirelessly. The identification of cancer stem-like cells (CSCs) or poorly differentiated tumors as targets of NK cells has finally shown the indispensable role these effectors play in successful cancer therapy. Since NK cells are responsible for elimination and differentiation of the aggressive CSCs/poorly differentiated tumors, it is logical that when the function of these cells is compromised, the patient could greatly be predisposed to tumor growth, expansion, invasion, and metastasis. In addition, by the aid of IFN-γ-mediated differentiation of tumor cells and increased expression of MHC class I, NK cells facilitate the targeting of differentiated tumors by CD8 + T cells. Moreover, NK cells are important in mediating antibody-mediated cellular cytotoxicity and lysis of oncolytic virus-infected tumor cells among other important functions.

This book consists of contributions from established leaders in the fields of NK and T cell biology and immunotherapy. The reviews address the latest advances in cancer immunotherapy using NK cells and T cells and highlight the challenges and difficulties in breaking the treatment-resistance of tumor cells surrounded by a suppressive tumor stroma that contain inhibitory immune factors. This book also reviews the current clinical applications of classical NK and T cell immunotherapies as well as the adoption of universal therapeutic NK and T cell preparations.

The book is separated into three sections. The first section summarizes the basics of cellular immunotherapies, delineating the basic immunobiology of NK cells, and differing roles of NK and T cells in targeting cancer. Also discussed is the intimate, synergistic interaction of NK cells and T cells and the role of NK cells in the expansion and functional activation of CD8 + T cells. Both CAR-NK and CAR-T therapeutics are now in many clinical trials, and their biology and significance in cancer treatments are also discussed in this section. Finally, the use of TILs in cancer treatment has been advocated by many investigators and is discussed in this section.

In Section I, Drs. Senjor, Jewett, and their colleagues discuss the basic biology of NK cells and their function in cancer as well as the impact of the tumor microenvironment (TME) on NK cell function. In addition, they introduce their novel engineering methodology for generating super-charged NK cells for use in tumor immunotherapy. Drs. Navin and Parihar describe different suppressive immune effectors within the TME and methods to overcome the suppressive nature of the TME. Dr. Matosevic discusses the use of NK cells in solid tumor treatments listing the advantages and the disadvantages of allogeneic NK cells in cancer therapy. Dr. Wu reviews the significance of NKG2D receptor signaling in NK activation and provides evidence for the use of this axis in successful immunotherapeutic strategies. In the chapter titled Chimeric antigen receptor-modified cells for the treatment of solid tumors: First steps in a thousand-mile March, Drs. Rafei, Basar, Rezvani, and Daher describe the successes and challenges of CAR-T therapy for hematological and solid tumors. Drs. Mahuron and Fong introduce TILs and discuss their significance in cancer therapy and the challenges encountered in the use of these cells in cancer therapy. Drs. Murad, Park, and Priceman discuss the status of current FDA-approved CAR-T cell products and their clinical impact, highlight unique hurdles for CAR-T cells for solid tumors, and provide perspectives on the future of CAR-engineered cell therapies.

In Section II, the authors present processes for trials optimization including novel diagnostics, assay readouts, influence of route of administration, and production optimization for cell therapy. Drs. Wang and Rivière delineate Optimization of production of cell therapies, including preclinical proof-of-concept studies, technology transfer, and process development. Drs. Volpe, Blasberg, Serganova, and Ponomarev discuss Imaging the immune cell in immunotherapy, providing a comprehensive summary of state-of-the-art imaging modalities for tracking of cancer immunotherapy. Drs. Feldman and Badie discuss the effective cell delivery systems for CNS cancers and list the challenges that cell therapists face when designing strategies to effectively target aggressive CNS tumors. Central to their discussion is the concept of regional delivery of cellular therapies that enhance their efficacy while offering the possibility of cellular therapy without the need for lymphodepletion. Finally, in the chapter titled Diagnostic methods to assess the numbers, phenotype and function of primary and engineered NK cells: Methods to predict prognosis and treatment outcome, Drs. Ko, Kaur, Chen, Breznik, Senjor, Chovatiya, Wong, Turnsek, Kos, and Jewett discuss novel diagnostic tests to assess the survival, expansion, and function of both primary NK cells and those that have been super-charged.

Section III is dedicated to reviewing trials and combinational strategies in cellular immunotherapy. Historically, most papers, books, and grant funding have been dedicated to only specific cell subsets. This has resulted in a wealth of knowledge for single cell type. However, it has fallen short in providing effective cancer therapeutics. We hope, going forward, academic focus and funding will be leveraged to understand how key immune effectors work in synergy. The key is to understand fully the multiple interactions between different immune cell subsets for the successful eradication of the tumors. The authors in this section present a number of important concepts and methodologies regarding patient trials and combinational strategies in cellular immunotherapy and indicate that successful cell therapy may depend on the selection of other treatment strategies synergistic with immune cell therapy. Drs. Sadeghi, Chen, Jewett, and Kaur discuss the differential targeting of undifferentiated vs well-differentiated tumors by the NK cells and combinatorial use of NK cells therapy with chemotherapy and radiation for maximum treatment efficacy. Drs. Chaurasiya, Woo, Choong, Deshpande, and Fong discuss the synergistic effect of oncolytic viral therapy with different cell therapies, listing the successes and challenges encountered in such therapies. Drs. Singh and Gumrukcu discuss different stages of cancer with its relevance in tailored therapeutic strategies and successful patient therapies using NK and T cells. Drs. Breznik, Novak, Majc, Habič, and Jewett discuss the existing trials and list the successes and challenges of NK cell therapy in glioblastoma and offer combinatorial treatments for the effective eradication of these aggressive tumors. Drs. Zhu, Bellis, Saini, Fong, and Adusumilli review the field of CAR-T therapy and provide future perspectives on the use of these cells in cancer therapy. Drs. Zeng, Li, Lee, and Yang discuss their novel NK T cell therapeutic strategies and provide the list of successes and challenges of using such cells in cancer immunotherapy. Finally, Drs. Maharaj, Polineni, Abreu, and Gouvea discuss Molecular remission using low-dose immunotherapy with minimal toxicities for poor prognosis hematological and solid tumor cancers.

The goals of this book are to review (1) the theory and practice of cancer cellular therapies; (2) the challenges and promises of efficient production, in vivo tracking of cellular therapies, and related biologic readouts; and (3) to summarize current trials data and the state of knowledge of combination therapies. This book is intended to summarize the field for current and future translational researchers at all levels. It is meant to be a guide for graduate students and postdoctoral fellows entering the field. It is meant to summarize the current state-of-the art for the investigators and trialists active in development and research in cellular therapies for cancer. Most of all, we are hoping this is one of the first clinical manuals for the emerging field of clinical cellular therapies for cancer. We hope that our audience of scientists, oncologists, radiologists, and colleagues in industry find this useful.

A work like this is only possible because of the contributions of many individuals. The authorship of this work includes scientific leaders in cell biology, cell engineers, trialists, cell production experts, and experts in molecular imaging. We thank them for their contributions and efforts to collaborate in the creation of this comprehensive and special work.

We also thank our teachers, graduate students, postdoctoral fellows, and colleagues who have shared their knowledge and experience with us. We thank our patients who inspire us to be superior clinicians and constantly strive to improve the field. We thank our Assistant Editor at Elsevier, Samantha Allard, for her efforts in keeping us on track and on time. Finally, we thank our families, especially our spouses Keith and Nicole, for the patience and support they have given us daily for our scientific work and then to complete this scientific book.

Anahid Jewett

Yuman Fong

Contributors

Maria M. Abreu     Institute for NeuroImmune Medicine, Nova Southeastern University, Ft. Lauderdale, FL, United States

Prasad S. Adusumilli

Thoracic Service, Department of Surgery

Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Behnam Badie     Division of Neurosurgery, City of Hope National Medical Center, Duarte, CA, United States

Rafet Basar     Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, United States

Rebecca Bellis     Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Ronald Blasberg

Department of Radiology

Department of Neurology

Molecular Pharmacology and Chemistry Program

Memorial Sloan Kettering Cancer Center, New York, NY, United States

Barbara Breznik

Division of Oral Biology and Medicine, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, University of California Los Angeles (UCLA), Los Angeles, CA, United States

Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia

Shyambabu Chaurasiya     Department of Surgery, City of Hope Comprehensive Cancer Center, Duarte, CA, United States

Po-Chun Chen     Division of Oral Biology and Medicine, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, University of California Los Angeles (UCLA), Los Angeles, CA, United States

Kevin Choong     Department of Surgery, City of Hope Comprehensive Cancer Center, Duarte, CA, United States

Nishant Chovatiya     Division of Oral Biology and Medicine, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, University of California Los Angeles (UCLA), Los Angeles, CA, United States

May Daher     Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, United States

Supriya Deshpande     Department of Surgery, City of Hope Comprehensive Cancer Center, Duarte, CA, United States

Lisa Feldman     Division of Neurosurgery, City of Hope National Medical Center, Duarte, CA, United States

Timothy D. Folsom

Department of Pediatrics

Masonic Cancer Center

Center for Genome Engineering

Stem Cell Institute, University of Minnesota, Minneapolis, MN, United States

Christina Fong     Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Yuman Fong     Department of Surgery, City of Hope Comprehensive Cancer Center, Duarte, CA, United States

Jacqueline Gouvea     The Maharaj Institute of Immune Regenerative Medicine, Boynton Beach, FL, United States

Anamarija Habič

Department of Genetic Toxicology and Cancer Biology, National Institute of Biology

Jozef Stefan International Postgraduate School, Ljubljana, Slovenia

Anahid Jewett

Division of Oral Biology and Medicine, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, University of California Los Angeles (UCLA)

The Jonsson Comprehensive Cancer Center, UCLA School of Dentistry and Medicine, Los Angeles, CA, United States

Sharyn I. Katz     Division of Thoracic Radiology, Department of Radiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States

Kawaljit Kaur     Division of Oral Biology and Medicine, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, University of California Los Angeles (UCLA), Los Angeles, CA, United States

Meng-Wei Ko     Division of Oral Biology and Medicine, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, University of California Los Angeles (UCLA), Los Angeles, CA, United States

Janko Kos

Department of Biotechnology, Jožef Stefan Institute

Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

Derek Lee     Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles (UCLA), Los Angeles, CA, United States

Zhe Li     Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles (UCLA), Los Angeles, CA, United States

Emil Lou

Masonic Cancer Center, University of Minnesota

Department of Medicine, Division of Hematology, Oncology, and Transplantation, Minneapolis, MN, United States

Dipnarine Maharaj     The Maharaj Institute of Immune Regenerative Medicine, Boynton Beach, FL, United States

Kelly Mahuron     Department of Surgery, City of Hope Comprehensive Cancer Center, Duarte, CA, United States

Bernarda Majc

Department of Genetic Toxicology and Cancer Biology, National Institute of Biology

Jozef Stefan International Postgraduate School, Ljubljana, Slovenia

Sandro Matosevic     Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN, United States

Branden S. Moriarity

Department of Pediatrics

Masonic Cancer Center

Center for Genome Engineering

Stem Cell Institute, University of Minnesota, Minneapolis, MN, United States

John P. Murad     Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, CA, United States

Ishwar Navin

Department of Immunology and Microbiology, Baylor College of Medicine

Center for Cell and Gene Therapy, Texas Children’s Hospital, Houston Methodist Hospital, and Baylor College of Medicine, Houston, TX, United States

Metka Novak     Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia

Robin Parihar

Department of Immunology and Microbiology, Baylor College of Medicine

Center for Cell and Gene Therapy, Texas Children’s Hospital, Houston Methodist Hospital, and Baylor College of Medicine

Section of Hematology/Oncology, Department of Pediatrics, Texas Children’s Hospital, Houston, TX, United States

Anthony K. Park     Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, CA, United States

Vineet Polineni     The Maharaj Institute of Immune Regenerative Medicine, Boynton Beach, FL, United States

Vladimir Ponomarev

Department of Radiology

Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Saul J. Priceman     Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, CA, United States

Hind Rafei     Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, United States

Jamie Rand     Department of Surgery, City of Hope Comprehensive Cancer Center, Duarte, CA, United States

Katayoun Rezvani     Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, United States

Isabelle Rivière

Cell Therapy and Cell Engineering Facility

Center for Cell Engineering

Molecular Pharmacology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, United States

Leonid Roshkovan     Division of Abdominal Imaging, Department of Radiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States

Saeed Sadeghi     Division of Hematology- Oncology, Department of Medicine, UCLA David Geffen School of Medicine, Los Angeles, CA, United States

Jasmeen Saini     Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Emanuela Senjor

Division of Oral Biology and Medicine, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, University of California Los Angeles (UCLA), Los Angeles, CA, United States

Department of Biotechnology, Jožef Stefan Institute

Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

Inna Serganova

Department of Neurology, Memorial Sloan Kettering Cancer Center

Department of Medicine, Division of Hematology and Medical Oncology, Weill Cornell Medicine

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Timothy K. Starr

Masonic Cancer Center, University of Minnesota

Department of Obstetrics and Gynecology and Women’s Health, Minneapolis, MN, United States

Tamara Lah Turnsek     Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia

Alessia Volpe     Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Xiuyan Wang

Cell Therapy and Cell Engineering Facility

Center for Cell Engineering

Molecular Pharmacology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, United States

Beau R. Webber

Department of Pediatrics

Masonic Cancer Center

Center for Genome Engineering

Stem Cell Institute, University of Minnesota, Minneapolis, MN, United States

Paul Wong     Division of Oral Biology and Medicine, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, University of California Los Angeles (UCLA), Los Angeles, CA, United States

Yanghee Woo     Department of Surgery, City of Hope Comprehensive Cancer Center, Duarte, CA, United States

Jennifer Wu     Feinberg School of Medicine, Northwestern University, Chicago, IL, United States

Lili Yang

Department of Microbiology, Immunology, and Molecular Genetics

Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research

Jonsson Comprehensive Cancer Center, David Geffen School of Medicine

Molecular Biology Institute, University of California Los Angeles (UCLA), Los Angeles, CA, United States

Yuan Yuan     Department of Medical Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, United States

Samuel Zeng     Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles (UCLA), Los Angeles, CA, United States

Amy Zhu     Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Section I

Basics of cellular immunotherapy: Differing roles of NK and T cells in targeting cancer and their intimate synergistic interaction underscores the requirement for the use of both cell types in successful cancer treatment

Chapter 1: Multifaceted nature of natural killer cells: Potential mode of interaction and shaping of stem cells

Emanuela Senjora,b,c; Meng-Wei Koa; Kawaljit Kaura; Po-Chun Chena; Barbara Breznika,d; Nishant Chovatiyaa; Janko Kosb,c; Anahid Jewetta,e    a Division of Oral Biology and Medicine, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, University of California Los Angeles (UCLA), Los Angeles, CA, United States

b Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia

c Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

d Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia

e The Jonsson Comprehensive Cancer Center, UCLA School of Dentistry and Medicine, Los Angeles, CA, United States

Abstract

Natural killer (NK) cells are the key immune effectors with the ability to lyse stem-like/poorly differentiated tumor cells and mediate differentiation through their cytotoxic activity and via secretion of IFN-γ and TNF-α, respectively. In this book chapter, we describe the basic biology of NK cells and their function in cancer as well as the impact of the tumor microenvironment on NK cell function. Furthermore, we present a novel strategy for the expansion of allogenic NK cells, which results in the production of large numbers of highly functional supercharged NK cells. Moreover, we discuss the recent advances in the application of NK cells in cancer immunotherapy. The combination of standard anticancer treatments with recently established immunotherapeutic approaches with NK cells should provide the optimal conditions for the eradication of tumors.

Keywords

NK cells; Cancer stem cells; Cancer immunotherapy; Supercharged NK cells; Differentiation; IFN-γ

Abbreviations

ADCC 

antibody-dependent cellular cytotoxicity

BiKE 

bispecific killer cell engager

CAR 

chimeric antigen receptors

CCL 

chemokine (C-C motif) ligand

CD 

cluster of differentiation

COX2 

cyclooxygenase 2

CSCs 

cancer stem-like cells

CTLA-4 

cytotoxic T-lymphocyte-associated protein 4

DC 

dendritic cell

EGFR 

epidermal growth factor receptor

GFP 

green fluorescent protein

GM-CSF 

granulocyte-macrophage colony-stimulating factor

Hu-BLT 

humanized-bone marrow/liver/thymus

IDO 

indoleamine 2,3-dioxygenase

IFN 

interferon

IL 

interleukin

ILC 

innate lymphoid cells

iPSCs 

induced pluripotent stem cells

KIR 

killer cell immunoglobulin-like receptors

LTi cells lymphoid-tissue inducer cells

MDSC 

myeloid derived suppressor cell

MHC 

major histocompatibility complex

NF-κB 

nuclear factor kappa B

NK cell natural killer cell

NSG mouse NOD SCID gamma mouse

OCs 

osteoclasts

OV 

oncolytic virus

PAMP 

pathogen-associated molecular pattern

PD-1 

programmed cell death protein 1

PGE 

prostaglandin E

TGF 

transforming growth factor

TIGIT 

T cell immunoglobulin and ITIM domain

TIM-3 

T cell immunoglobulin and mucin-domain containing-3

TLR 

Toll-like receptor

TME 

tumor microenvironment

TNF 

tumor necrosis factor

TRAIL 

TNF-related apoptosis-inducing ligand

TriKE 

tri-specific killer cell engager

Acknowledgments

The authors are grateful to numerous undergraduate and graduate students, post-doctoral fellows, visiting scholars, and faculty members for their excellent contributions to the work presented in this chapter. In addition, we thank our funding agencies and donors for supporting the work.

Conflict of interest

No potential conflicts of interest were disclosed.

Natural killer cells: Overview and background

Natural killer (NK) cells differ morphologically and phenotypically from T and B lymphocytes and were defined traditionally as cells capable of spontaneously killing tumor cells [1]. NK cells are defined as large granular lymphocytes [2], which comprise 5%–20% of the peripheral blood lymphocytes [3]. NK cells are generated in the bone marrow and are crucial in lysing virally infected cells as well as malignantly transformed targets [4]. They are defined by the surface expression of CD56 [5] and CD16 [6] and lack the expression of CD3 surface receptors [7]. CD16 receptors, also known as FcγRIII receptors, expressed on the surface of NK cells constitute one of the main receptors on NK cells important in mediating both direct cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) [8,9]. In addition to circulating NK cells, many are also found in different tissues, such as lymphoid organs, healthy skin, gut, liver, lung, and uterus to name a few [10]. The missing-self hypothesis proposed in 1986 speculated that the presence of MHC class-I on the surface of the NK-interacting cells is responsible for the inhibitory signals delivered to the NK cells [11]. Since then many inhibitory receptors, such as NKG2A [12], p58 [13], KIR2D [14], and activating receptors, such as NKp46 [15], NKp30 [16], NKp44 [17], and NKG2D [18], have been defined. The net sum of activating and inhibitory signals that NK cells receive determines the levels of NK cell activation. The threshold for activation of NK cells is lowered in a suitable cytokine microenvironment provided by IL-2, IL-12, IL-15, IL-18, and type I interferons [19].

NK cells exhibit two main or primary effector functions: cytotoxicity and release of cytokines (Fig. 1). NK cells mediate cytotoxicity via two different mechanisms: granule-mediated cytotoxicity and cytotoxicity mediated by the death receptors. Release of cytotoxic granules induces both the necrotic and the apoptotic cell death via the activation of caspases. The granules contain perforin, a calcium-dependent pore forming protein, and serine peptidases known as granzymes [20]. Both perforin and granzymes are synthesized in their precursor forms and are required to be cleaved by cathepsins to their active forms. Processing of perforin is catalyzed by cathepsin L [21], whereas cathepsins C and H activate the granzymes A and B [22–24]. The activity of these cathepsins is further regulated by cystatin F [25]. NK cells express ligands for death receptors, such as Fas ligand, TNF-α, and TRAIL (Apo2L). Upon interaction with the death ligands on NK cells, the target cells receive signals to undergo cell death [26]. Both pathways can also be activated via ADCC induced by the binding of Fc portion of the antibody on target cells to CD16 receptors on NK cells [27].

Fig. 1

Fig. 1 Roles of NK cells in cancer.NK cells have different roles in cancer. In the tumor microenvironment, they are able to eliminate CSCs by three different mechanisms: granule-dependent cytotoxicity, death receptor-mediated cytotoxicity, and antibody-dependent cellular cytotoxicity. In addition to eliminating CSCs by killing, they eliminate CSCs by differentiation caused by released cytokines, especially IFN-γ. Finally, NK cells help in the activation of CD8+ T cells. After CSCs are killed, tumor antigens are released. Secreted IFN-γ is important for the differentiation and activation of dendritic cells. Dendritic cells present tumor antigens to CD 4 + T cells, which leads to activation of CD8+ T cells. NK cells can act synergistically with various cancer treatment strategies. Chemotherapy and radiotherapy are usually more efficient in eliminating differentiated cancer cells. Treatment with NK cells would be beneficial in eliminating the remaining CSCs. NK cells also mediate their cytotoxicity through ADCC. By designing antibodies that bind to the CD16 receptor on NK cells and tumor-specific antigens, we can bring together effector cells and target cells. In addition, therapeutic antibodies can be further engineered to express supportive cytokines and matrix metalloproteinase inhibitors or inhibit immune checkpoints. Oncolytic viruses also represent a good treatment option as they switch the tumor environment from immunologically cold to immunologically hot, allowing immune cells to function.

Upon activation, NK cells secrete an array of cytokines, chemokines, and growth factors, among which IFN-γ is one of the key cytokines [28]. IFN-γ enables the activation of dendritic cells (DCs) and monocytes. This mechanism is important for the activation and expansion of adaptive immune responses [29,30]. In addition, IFN-γ produced by the NK cells stimulates the expression of MHC class-I and -II molecules on the cell surface of their interacting cells [31], mediating inhibition of tumor proliferation [32], and upregulation of adhesion molecules, which increases the sensitivity of tumors to CD8 + T cell cytotoxic effectors [33] (Fig. 1).

NK cells were found to have specific targets, as they were only able to kill certain tumor cells, but not those highly susceptible to T cell killing [34]. It was later discovered that they preferentially targeted cancer stem-like cells (CSCs)/poorly-differentiated tumor cells that expressed lower levels of MHC class-I molecules on their surface [35–38] (Fig. 1).

NK cells form part of the recently discovered family of innate lymphoid cells (ILCs). Latest agreement on nomenclature divides ILCs into NK cells, ILCs type 1, 2, and 3 (also referred to as helper ILCs), and lymphoid-tissue inducer cells (LTis) [39]. ILCs do not have antigen-specific receptors, but they do mirror the function of T lymphocytes. ILC1s respond to intracellular pathogens, similar to CD4 + Th1 cells. ILC2s, similar to CD4 + Th2 cells, react to extracellular parasites and allergens, while ILC3s react to extracellular bacteria and fungi [40]. By that analogy, NK cells are the innate counterparts of CD8 + T cells. In contrast to T lymphocytes, the time of action of ILCs is at an early phase in the immune response [41].

Although all ILCs are developed in the bone marrow, ILC1, 2, and 3 are localized in mucosa-associated tissues, whereas NK cells are localized in the blood stream and secondary lymphoid organs. As helper ILCs are mainly tissue resident, they are in an ideal location to fine-tune antitumor immunity [42]. While NK cells undergo constant replenishment from the bone marrow, other ILCs remain in the tissues throughout life and are replenished by local proliferation of tissue resident progenitor cells [43]. Among the ILCs, only NK cells have cytotoxic properties against virally and malignantly transformed cells. Some reports indicate that ILC1s are weakly cytotoxic, but mainly they represent the first line of defense against some viruses and bacteria [44,45]. ILC2s mediate the response to extracellular helminth parasites, while ILC3s are important for maintaining tolerance to symbiotic microbiota [42]. Both ILC2 and 3 can present antigens via MHC class-II, but ILC3 do not possess necessary costimulatory factors [46,47]. LTis are important in the fetal developmental stage as they induce the formation of secondary lymphoid organs and Peyer’s patches [48]. Subsets of ILCs are identified by differentially expressed surface receptors and by the secreted cytokines. Both NK cells and ILC1s secrete IFN-γ and TNF-α, while ILC2s secrete type 2 cytokines (IL-4, IL-5, IL-9, IL-13, and amphiregulin), and ILC3s and LTis secrete IL-17, IL-22, and GM-CSF [42].

The role of ILCs in tumor defense differs depending on the tumor type and the microenvironment [49]. Depending on the signals from the microenvironment, they can convert from one subset to another [50–52]. NK cells in their fully functional state are able to eliminate the most resistant CSCs/poorly differentiated tumors. Under the influence of TGF-β, NK cells can be converted to intermediate ILC1s, which do not have antitumor properties. Helper ILCs were shown to promote immunosuppressive microenvironment in cancer patients. Different factors, such as increased CTLA-4 and GM-CSF, low IFN-γ levels, CCL5, and TGF-β, contribute to the pro-tumoral microenvironment [51,53]. ILC2s, which produce suppressive IL-5 and IL-13 cytokines [54], are found to be increased in gastric [55], prostate [56], and breast cancers [57]. In addition, ILC2s produce amphiregulin that promotes EGFR expressing tumor cells leading to tumor invasion and metastasis [58]. ILC3s promote tumorigenesis of colorectal cancers by secretion of IL-22 and IL-17 [59]. In breast cancer, increased numbers of ILC3s correlated with the increased metastasis to lymph nodes [60]. In contrast, other reports suggested a beneficial role for ILC3s in cancer. In B16 melanoma mouse model, ILC3s were able to recruit CD8 + T cells and NK cells to the tumor site [61]. Future studies should be directed to defining the exact similarities and differences between ILCs and those of other traditionally defined immune effectors.

Two faces of NK cells: Concept of split anergy in NK cells and its potential role in tumor differentiation

NK cells enter various stages of development after their generation in the bone marrow. Subsequent to generation of NK cells, the licensing takes place, which selects for NK cells expressing MHC class-I receptors and are therefore thought to be tolerant to self [62]. Two subsets of NK cells have been identified based on surface expression of CD56 and CD16. The minor subset makes up approximately 10% of human NK cells and is defined as CD56brightCD16dim. This subset is speculated either to be a less mature form of NK cells or to develop from the CD56dimCD16bright subtype. This subtype is able to secrete cytokines, but possesses no or low cytotoxic ability. Fully functional cytotoxic and cytokine releasing CD56dim and CD16bright NK cells represent the major subset of NK cells in humans (about 90%), also defined as stage I NK cells [27]. After interaction with target CSCs or activation with IL-2 and CD16 monoclonal antibodies, NK cells enter in stage II and become split anergized cells, exhibiting the phenotype of CD16lowCD56brightCD69bright. The cytotoxic ability of split-anergized NK cells is low, but they secrete higher levels of cytokines such as IFN-γ and TNF-α [63–65]. In the absence of rescue signals, NK cells enter stage III and lose the ability to secrete IFN-γ and TNF-α. Instead, these NK cells will likely secrete cytokines such as IL-6 and IL-10, similar to those seen by NK92 cell line [66]. Finally, NK cells enter stage IV and become unresponsive, exhausted, and eventually undergo senescence and apoptosis (Fig. 2) [67]. Even though split anergized NK cells lose their cytotoxicity, they are still important in the tumor microenvironment (TME). Owing to their secretion of IFN-γ, split anergized NK cells contribute to the differentiation of CSC/poorly differentiated tumors by increasing MHC class-I, CD54, and B7H1 (PD-L1) and decreasing CD44 surface expression levels on tumor cells (Fig. 1). Differentiated tumors are known to be susceptible to T cell-mediated killing [68–70]. In addition, differentiated tumor cells have a lower ability to proliferate and metastasize [63,71,72]. Overall, these results indicated that NK cells are important in shaping the TME.

Fig. 2

Fig. 2 Stages of NK cell phenotype and function.Peripheral blood NK cells mediate cytotoxicity against CSCs and secrete cytokines, especially IFN-γ (stage I). Cytotoxic granules containing granzyme B and perforin facilitate one of the possible cytotoxic pathways. After interaction with CSCs or in vitro activation with IL-2 and anti-CD16 monoclonal antibodies, NK cells become split-anergized and enter the stage II. Split-anergized NK cells are no longer cytotoxic, but they have higher cytokine secretion, which allows differentiation of CSC to more differentiated phenotypes. Without rescue signals, the cells enter stage III and become nonfunctional and harmful in the TME as they secrete IL-6 and IL-10. Eventually, NK cells become apoptotic and enter stage IV. We can expand supercharged NK cells from split anergized cells. By coculturing split anergized NK cells with monocyte-derived osteoclasts expressing NKG2D ligands (ULBPS and MICA/B) and cytokines IL-12, IL-15, IL-18, and IFN-α, with the addition of sonicated probiotic bacteria sAJ2, we can produce highly cytotoxic supercharged NK cells with a higher ability to secrete cytokines and a higher proliferation potential.

The rationale for the functional activation of NK cells in many gene knockout mice: Potential common mechanism of activation

NK cells are shown to be activated in many knock-out or knock-down mice (Fig. 1). We have previously reported a comprehensive list of genes that upon deletion are implicated in the triggering of augmented NK cell function in in vitro culture conditions and in mice models [73]. For example, blockage of NF-κB in oral tumor cells not only augmented the proliferation and activation of NK cells but also of CD8 + T cells [74]. Furthermore, the secretion of immunosuppressive cytokine, IL-6, was decreased, which benefited the activation of NK cells. Moreover, the knockdown of NF-κB downregulated the expression of MHC class-I on Hep2 tumor cells, rendering these tumors more susceptible to NK cell-mediated killing [75]. Similarly, knock down of COX-2 in nontransformed healthy myeloid cells and mouse embryonic fibroblasts augmented NK cell function and expansion [76]. Expression of CD44, a cell surface glycoprotein that facilitates binding of NK cells to tumor cells, correlates with