Engineering NK Cells Towards Next Generation NK Cell Immunotherapy

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Engineering NK Cells Towards Next Generation NK Cell Immunotherapy

By

Vincent OEI Yi Sheng

㜑㢪㜌

Department of Cancer Immunology Institute for Cancer Research

at

The Norwegian Radium Hospital Oslo University Hospital

Faculty of Medicine University of Oslo

Oslo, 2018

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© Vincent OEI Yi Sheng, 2018

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8377-280-7

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

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Acknowledgements

The work presented in this thesis was carried out at the Department of Cancer Immunology, Institute for Cancer Research, at The Norwegian Radium Hospital, Oslo University Hospital from November 2012 to June 2017. The bulk of the financial support comes from Stiftelsen Kristian Gerhard Jebsen that has donated generously to set up the K.G. Jebsen Centre for Cancer Immunotherapy under the Leadership of our Department Leader Professor Johanna Olweus. This thesis thus seeks to achieve the listed goals of the centre. I also would like to acknowledge the administrative support of the University of Oslo with the centre.

Furthermore, I would like to express my dearest gratitude to the following wonderful and amazing people:

My supervisor Professor Karl-Johan Malmberg for his amazing leadership providing latitude and breath to allow everyone to pursue our goals under his direction. It has been an honour to join him in starting the laboratory in Oslo from scratch to such a well-oiled machine that has begun to show ground-breaking results. He is an excellent role-model of a Clinician- Scientist that I will love to emulate in the years to come to be able to communicate and reach out to many people for collaboration and achieve a greater goal. He has help me mature to be a better person.

My co-supervisor Dr Jodie (Jode) Peter Goodridge for his amazing zen qualities that brings stability to the group and made me a better person in life. His approach to handle situations has been valuable in moulding my character in Science. His support, advice, scientific inputs and guidance are invaluable to me. His great ideas, innovation and insight into matter beyond my conception has push me into a new arena beyond myself and will continue to inspire me in the many years to come. I will also miss the many late nights we shared and the “buffy coat gang” we formed that created great science over conversations and sometimes a beer.

My laboratory administrators/engineers Hanna Julie Hoel and Dr Merete Wiiger for their “mothering” nurture of everyone in our laboratory. They ensure that the group runs smoothly and provided many support for our research that has ensure the success of our work. I thanked Julie for the company and the welcome in the first year in Oslo and the memorable times we had when we were young. Also, I thank Merete for her constant guidance and support to take over many of the task I have accumulated during the start-up of the laboratory.

The buffy coat guys and the late-nighters in the laboratory apart from Jode, Dr Benedikt Jacobs, Dr Yang Weiwen, Dennis Clement, and Stanley Cheuk. They kept me company in the evenings and provided valuable scientific inputs and moral support. They made the laboratory feel like a home away from home.

The entire members of the Malmberg group in Oslo and Stockholm, Andreas, Vivien, Lisa, Aline, Ebba, Alvaro, Astrid, Trevor, Eivind, Michelle, Kishan, Axel, Daniel and many more. Thank you for the wonderful environment you have created to make this group a success and amazing. I would also extend my gratitude to my Polish colleagues Dr Martha and Dr Agneizska for their kind support to make this effort a success. Their efforts and collaboration to help me with the loose ends of the paper when I am in Sinagpore has made this thesis a success and I am eternally grateful for that.

My Department leader Professor Johanna Olweus who has been ever so supportive in all my endeavours to make the department a better place and all the challenges I faced internally and externally in all aspects of my life in Oslo. Her inspiring leadership has taught

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me a lot and will be very much applicable to my career in the future. I am so fortunate to have such an extensive support in my work and in science from her.

The members of Professor Olweus group. They are too many to list over the years and they have been ever so crucial to my success and their support has been keeping me going through all the challenges in Oslo. In particularly, Eirini for the wonderful time in our office, Dr Shraddha Kumari for the food and moral support, Maxi-Lu for the valuable feedback and support, and Dr Fan Ying for her guidance, language and moral education to make me a better person. I would also like to thank all the ladies (and Ali) for the support of the “Buffy Coat Gang/Guys” to share the samples to achieve high turnover with maximal utility of the resources available to us in Oslo. I hope for their continued support to ensure high quality usage of buffy coat for our research.

The entire department of Cancer Immunology for the feedback and scientific inputs that they have provided over the years. In particularly, Po Yong Fang for her silent and under- recognised support of the laboratory with supplies and for me a dear friend who I will value for the rest of my life. She has made life in Oslo and the laboratory so much more liveable.

I would also like to thank the Singaporeans in Oslo and back home for their facilitation and kindness to make this thesis possible. A special mention to Professor Helmer Aslaksen and his wife Karen Moh, Professor Kjell Kristoffersen and his wife Jan Yee are valuable friends who I treasure as they introduce me into the Norwegian life and make me part of their family so that I will not be alone in Oslo. Their kindness and hospitality shall be paid forward into the future and dearly remembered. I would also express gratitude to Dr Koh Shimin Grace for changing my life into this path that leads me towards my dream of becoming a Clinician-Scientist.

Finally, I would like to thank my family and friends in Singapore and Oslo. They have all made a difference in my life to make all these possible. Most of all, this thesis is dedicated to my mother Madam Yeo Chin Guik who raised me and my sister single-handedly and taught me the values that made me who I am. Her nurture, support, encouragement, discipline, toils and pains has brought me to where I am and beyond.

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My inspiration, guidance, dream, and goal

I NVENTAS VITAM JUVAT EXCOLUISSE PER ARTES

Vergilius Aeneid, the 6th song, verse 663.

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LIST OF PUBLICATIONS

This thesis is based upon the following publications and manuscripts, which are referred to in-text by their roman numerals:

I. Targeting B-cell neoplasia with T-cell receptors recognizing a CD20-derived peptide on patient-specific HLA.

Nadia Mensali, Fan Ying, Vincent Oei Yi Sheng, Weiwen Yang, Even Walseng, Shraddha Kumari, Lars-Egil Fallang, Arne Kolstad, Wolfgang Uckert, Karl Johan Malmberg, Sébastien Wälchli, Johanna Olweus

OncoImmunology 2016 Feb 18;5(5)

II. Ex Vivo Expanded Adaptive NK Cells Effectively Kill Primary Acute Lymphoblastic Leukemia Cells.

Lisa L. Liu, Vivien Béziat, Vincent Oei Yi Sheng, Aline Pfefferle, Marie Schaffer, Sören Lehmann, Eva Hellström-Lindberg, Stefan Söderhäll, Mats Heyman, Dan Grandér, Karl-Johan Malmberg

Cancer Immunology Research, 2017 Aug; 5(8): 654-665

III. Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors.

Vincent Oei Yi Sheng, Hanna Julie Hoel, Weiwen Yang, Hilde Almåsbak, Marta Siernicka, Agnieszka Graczyk-Jarzynka, Magdalena Winiarska, Radoslaw Zagozdzon, Johanna Olweus, Jon-Amund Kyte, Karl-Johan Malmberg

Cancer Immunology Research, Issue 6(4) April 2018. Accepted 1^st Feburary 2018 IV. Flow cytometry-based metrics of the educated state

Vincent Oei Yi Sheng, Dennis Clement, Benedikt Jacobs, Kishan Kumar Chudasama, Merete Thune Wiiger, Jodie Peter Goodridge, Karl-Johan Malmberg

Manuscript in preparation.

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LIST OF ADDITIONAL RELEVANT PUBLICATIONS NOT INCLUDED IN THE THESIS

These are relevant publications not included in the thesis but are essential in illustrating concepts in the thesis. They are referred to in-text by their Arabic numerals with a prefix “S”.

1. Harnessing adaptive natural killer cells in cancer immunotherapy

Lisa L. Liu, Aline Pfefferle, Vincent Oei Yi Sheng, Andreas T. Björklund, Vivien Béziat, Jodie P. Goodridge, Karl-Johan Malmberg

Molecular Oncology, Volume 9, Issue 10, December 2015, Pages 1904-1917

2. Critical Role of CD2 Co-stimulation in Adaptive Natural Killer Cell Responses Revealed in NKG2C-Deficient Humans

Lisa L. Liu, Johannes Landskron, Eivind H. Ask, Monika Enqvist, Ebba Sohlberg, James A. Traherne, Quirin Hammer, Jodie P. Goodridge, Stella Larsson, Jyothi Jayaraman, Vincent Oei Yi Sheng, Marie Schaffer, Kjetil Taskén, Hans-Gustaf Ljunggren, Chiara Romagnani, John Trowsdale, Karl-Johan Malmberg, and Vivien Béziat

Cell Reports, 2016;15(5):1088-1099.

3. Accumulation of Dense-core Granules Determines Functional Potential in Educated NK cells

Jodie P. Goodridge, Benedikt Jakobs, Trevor Clancy, Ellen Skarpen, Andreas Brech, Michelle L. Saetersmoen, Johannes Landskron, Aline Pfefferle, Merete Thune Wiiger, William E. Louch, Eivind Heggernes Ask, Lisa L. Liu, Vincent Oei Yi Sheng, Una Kjällquist, Sten Linnarsson, Kjetil Taskén, Harald Stenmark, Karl-Johan Malmberg Immunity, In revision.

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The immune system is like an organism’s defence forces, from simple molecules protecting a plant from its enemies to a complicated system of cells patrolling the organism for its entire lifetime in mammals. They are comparable to a nation’s border defence which can be as simple as being surrounded by water or a wall, a passive barrier like our skin. It can also be as complicated as a combined force of aerial, naval and land defence forces with sophisticated weapons systems, like antibodies suspended in the extracellular fluids, leukocytes flowing in the blood, and microglia in the brain. Humans has knowing/unknowingly been imitating biology for millennia and as our ability to defend our territory has improved tremendously with modern technologies, so have our ability to defend our body from threats within and outside with advances in technologies. Even then, the lessons we are learning from our immune systems is ever growing in complexity and volume.

Before humans created our own defence force to defend our territories, animals have been marking their territories with their own bodily fluids. Even before that, multicellular organisms have developed molecules to defend itself against threats from invaders such as toxins or defensins in some plants, invertebrates, and vertebrates, and lactoferrin in mammals. Ascending the phylogenetic tree of life, we began to observe ever more specialised and sophisticated form of defence in the form of receptors involved in intracellular detection and elimination of invaders and threats (toll-like receptors), specialised cells expressing receptors that seeks out threats based on molecular patterns (neutrophils, macrophages, etc), and specialised cells that adapts to the environment and threats that it has encountered (T/B cells). The immune system can be classified into two main segment:

innate and adaptive immune system. It is worth noting that the two systems are no longer well delineated with increasing overlap of functions across both systems^{1, 2}. The innate immune system includes both molecular and cellular defence which in general are evolutionally pre-defined, phylogenetically ancient and constitutively active. It is thus no surprise that they are highly conserved across multicellular organism kingdoms. The adaptive immune system is generally cellular in nature and they are developed much later phylogenetically in most vertebrates. The adaptive immune system seeks out molecular signals presented on specialised molecules on cell surface which activate the relevant cells to generate a specific counter response and memory to protect against a re-challenge. Such molecules are thus adeptly named antigens and presented on antigen presenting cell (APC).

Together, they defend the organism against numerous threats occurring at every moment of its lives.

There are several parallels between biology and human’s creation that can be drawn.

The physical barrier and border control (like a fort) holds intruders at bay just like our skin and molecular defences, eliminating obvious threats before they can establish themselves.

The innate immune cells are like our organic troops, patrolling the territory constitutively, seeking for danger or pathogen associated molecular markers for elimination. The adaptive immune cells are like innate cells, behaving like organic troops with more specialised and long lasting defence against specific threats. The monocytes are like recruits to the infantry and support forces with great potential to develop and mature into macrophages or dendritic cells. As macrophage, they function like infantry troops which patrol and guard the tissue/territory, eliminating threats according to presence of the right molecular triggers, repair infrastructures as well as providing some reconnaissance for their peers. As dendritic cells, they function like scouts and signallers to communicate information on threats to higher echelons and provide support to eliminate threats. Similarly, other granulocytes (neutrophils,

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eosinophils and basophils) are functioning like police and guards that can respond and support appropriate action against the enemy. In many modern militaries, officers, artillery forces, specialised forces are essential to secure the win over a battle. In our adaptive immune system, the B cells are like the artillery and air forces where they “fire” their ammunitions from afar against their targets with ever increasing precision through adaptation.

Similarly, the T cells are the officers and special forces where the CD4 (officers) mediate the attack strategy and the CD8 (special forces) carry directed assassination missions against threats in our body. In more advanced armies, they have another group of elite forces which is given more autonomy over their actions to infiltrate, evaluate and eliminate threats as it sees fits. These forces are given a set of pre-defined objectives on rules of engagement and expected act autonomously against threats promptly. In our body, these are the natural killer cells (NK). Armed with a set of objectives on rules of engagement, these cells can perform simple logic functions to evaluate the threat based on both danger/pathogen associated molecular patterns as well as irregular behaviours to perform the appropriate remedial actions whether to kill a cell or not. However, such elite forces are limited in numbers and liberty to ensure that they do not go rouge. During peacetimes, such troops are on guard but not actively engaging any threats unless required.

1.1BASIC CONCEPTS OF NK CELL BIOLOGY

NK cells were first described in the 1970s by Kiessling et al. and Herberman et al. for their ability to engage and lyse target cells without prior sensitisation^{3, 4, 5, 6}. Responsible for

“background killing” of tumour cells in their T cell cytotoxicity assays, these cells were given their namesake of natural killer cells. In the 1980s, inspired by the Swedish naval public announcement to recognise their own submarines in defence against foreign submarines intrusion, the missing-self hypothesis was postulated by Klas Kärre to explain how NK cells recognise their target for engagement in a similar manner. This is counter-intuitive to contemporary concepts of immune response then where immune cells (both innate and adaptive) are supposed to respond to foreign antigens through their receptors. In this case, NK cells should behave like a T cells but independent of the peptide presented on MHC class I on the target. However, this paradigm shift was supported by hybrid resistance phenomenon in mice where a crossed progeny (AxB)F1 hosts rejected bone marrow or lymphoma grafts from both parental strains despite absence of any foreign antigen⁷. Therefore, NK cells’

function complement that of a T cells by targeting cells that attempted to evade T cell immunity by downregulating their MHC class I molecules⁸. This breakthrough opened up the possibility of an immune cell recognising its target by loss of self-ligand (termed “missing- self”) ⁸, and conversely a constitutive inhibition to establish tolerance.

The ability to recognise self-ligand over antigen also explains some of the receptor properties of NK cells. Despite being a lymphocyte with many similarities with T cells, NK cells do not undergo receptor recombination to generate large variety of receptors for antigen specificity^{9, 10} nor do they show clear evidence of immune memory until recently11, 12, 13, 14, 15. Therefore, NK cells are generally considered to be part of the innate immune system where their function is mediated by its pre-defined receptors. In many mammalian species including human, NK cells can be defined as CD3-NKp46+ lymphocytes as NKp46 is the most reliable marker for NK cells across most mammalian species. However, CD56 is another marker for NK cells in human which has an advantage of dividing NK cells into two subgroups based on its expression level. CD56bright NK cells are typically less mature with robust cytokine secretion profile but minimal cytotoxicity¹⁶. CD56dim NK cells are more mature with a great diversity of cytotoxic function within the subset with diminishing cytokine secreting function¹⁶.

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The frequency of NK cells in peripheral blood mononuclear cells typically range from 5-25% and can be found in many organs (liver, uterus, lungs, and gut) as resident cells, each with their unique phenotype markers and function^{17, 18}. Typically, NK cells are essential for killing virus infected cells, immunesurveillance against tumour cells^{19, 20}, placentation and immunesurveillance during pregnancy^{21, 22}. NK cells perform its cytotoxicity function either by release of cytoplasmic granules containing perforin and granzymes into the immune synapse upon direct recognition of the target or induction of apoptosis through FAS/FAS-L and TRAIL interaction²³. Since the postulation of missing-self hypothesis, NK cell recognition of target has progressed with increased complexity. One way of recognising its target is through interaction between its CD16 (FcγRIII) with IgG antibody during antibody dependent cellular cytotoxicity^{23, 24, 25}. NK cells can also be activated by a loss of self-ligand⁸ with/without presence of activating ligands which will be elaborated in the next section.

1.1.1 NK CELL RECEPTORS

Figure 1. Mechanisms of NK cell activation and response. MHC class I (inhibitory ligand) is expressed in most normal cells to commonly inhibit NK cells and ensure tolerance in the presence or absence of stimulatory ligands. When a cell is transformed or virally infected, it loses its expression of NK cell inhibitory ligand (missing self) or increase expression of the stimulatory ligands (induced self) or both, becoming the target of NK cells. This leads to the activation of NK cells to respond and kill the target.

NK cell response is mediated by a logic sum function created by an interplay between multiple activating and inhibitory signals as described in Figure 1. As shown in Figure 1, an NK cell recognises a cell as its target if it expresses increased ligand for NK cell stimulatory receptor (such as CD155) or loses expression of major histocompatibility complex (MHC) class I molecule which engages with NK inhibitory receptors. The actual algorithm has not been determined although inhibitory interactions via killer-cell immunoglobulin-like receptors (KIRs) and NKG2A, whose ligands are lost in missing-self response, is more dominant to maintain tolerance to self. In humans, this is mediated by KIRs^{26, 27}. In mice, the corresponding receptors are Ly49 receptors^{28, 29}. Both receptors recognise MHC class I of their cognate species and have analogous function in their host but are independently evolved in each species as two sets of unrelated receptors³⁰.

In humans, the KIR genes locus is at chromosome 19, part of the Leukocyte Receptor Complex (LRC)³¹. The structure of the receptor dictates its nomenclature. The first half of the name is determined by the number of its extracellular immunoglobulin-like domains (2D=two, 3D=three) followed by a letter denoting the length of its cytoplasmic tails (L=long S=short)

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and a number denoting the order of description. KIRs with short cytoplasmic tails tend to associate with immunoreceptor-tyrosine-based activating motifs (ITAM) on DAP12 signalling molecule, generating an activating signal³². KIRs with long cytoplasmic tails tend to associate with immunoreceptor-tyrosine-based inhibiting motifs (ITIM), generating an inhibitory signal³³. There are exceptions to the above trend but their actual function remains to be defined since they have yet to show any major role in regulating NK cell response.

Furthermore, not all natural ligands for all 15 known KIRs have been discovered yet and new KIRs are still being found. However, allelic variants of Human Leukocyte Antigen (HLA, synonym for human MHC)-A, B, C has been identified as ligands for up to 9 of them³⁴, summarised in Table 1. It is worth mentioning the ligands for KIR2DL1/2/3, KIR2DS1 and KIR3DL1 here. The ligands for KIR2DL1 and KIR2DL2/L3 are HLA-C with either lysine or asparagine at position 80 (labelled HLA-C2 or HLA-C1) respectively³⁵. HLA-C2 is also the ligand for activating KIR2DS1³⁵. The ligands for KIR3DL1 are Bw4 motifs at position 77-83 on HLA-A and B³⁶.

Table 1 – NK cell receptors and their ligands (where defined)

The KIR gene locus can be divided into two haplotypes based on the combination of genes inherited: A and B. KIR 3DL3, KIR2DL4 and KIR3DL2 are framework genes³⁷ common to both haplotypes and denoting the centromeric and teleomeric regions of the locus respectively³⁸. Haplotype A is the most common haplotype consisting of three inhibitory KIRs (2DL1, 2DL3 and 3DL1) and 2DS4. Haplotype B is made up mainly of a diverse combination of the remaining genes, mostly activating KIRs, sometimes with some of the haplotype A KIRs^37,

39. The inheritance of KIR genes is further complicated by random segregation, cross over across framework genes and independent assortment into at least 16 combination sets³⁷. However, selection pressure upon different human populations resulted in an assortment of stable haplotype frequencies among them^{40, 41, 42}. Resistance to viral infection comes at the price of procreation woes^{43, 44, 45}. A good illustration of that are people with A/A haplotype and HLA C1 homozygosity where the resistance to hepatitis C and Ebola viral infection comes at the elevated risk of complications of pregnancy and low birth weight if the foetus inherited HLA-C2 from the father^{43, 44, 45}. The greater

Receptors Ligands Inhibitory Receptors

KIR2DL1 HLA-C2

KIR2DL2/L3 HLA-C1

KIR3DL1 HLA-Bw4 ⁸⁰I, HLA-Bw4 ⁸⁰T

KIR3DL2 HLA-A3, HLA-A11, HLA-F

NKG2A HLA-E ILT2 (LIR-1) HLA- class I, UL18 Activating Receptors

KIR2DS1 HLA-C2

KIR2DS2 Not defined

KIR2DS3 Not defined

KIR2DS4 HLA-A11, some alleles of

HLA-C

KIR2DS5 Not defined

KIR3DS1 HLA-Bw4, HLA-F

NKG2C HLA-E

NKG2D ULBP1-4, MICA, MICB

CD2 CD58 (LFA-3)

CD16 IgG

DNAM-1 PVR, Nectin-2

NKp30 B7-H6, BAT-3, HSPG

NKp44 Hemagglutinin

NKp46 Hemagglutinin, HSPG

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missing-self response during viral infection comes at the price of inadequate placentation due to inhibitory interactions between HLA-C2 and 2DL1 in the uterus³⁰.

These inhibitory interactions prevent sufficient trophoblast invasion for spiral artery formation²¹. The converse applies where activating KIRs in haplotype B individuals receives protection from such complication and achieve better placentation during pregnancy²¹ but less likely to survive Ebola infection⁴⁵. Furthermore, haplotype B donors are also associated with improved outcomes in several studies on allogenic hematopoietic stem cell transplantation (HSCT) against ALL and AML46, 47, 48, 49. Therefore, the KIR repertoire is an important consideration in human health and design of NK cell based immunotherapy.

Another family of NK cell receptor crucial in NK cell biology is the C-type lectin receptors on chromosome 12 in the natural killer complex (NKC) gene cluster encoding for NKG2A, NKG2C and NKG2D^{50, 51}. They are also listed in Table 1. With ITIM in the cytoplasmic domain, NKG2A is the only inhibitory receptor among the three receptors of significance in the NKG2 locus⁵². NKG2C is an activating receptor that interacts with DAP12 to initiate an activation signal^{53, 54}. NKG2D, expressed as a homodimeric receptor on immune cells⁵⁵ including NK cells, initiates stimulatory signal with DAP10 in NK cells⁵⁴. NKG2A, NKG2C, and together with other lesser known NKG2 receptors all form heterodimer with CD94 which can interact with their common ligand, HLA-E, a non-classical MHC class Ib antigen⁵⁶. When co-expressed in vivo, NKG2A has a stronger affinity for HLA-E than NKG2C and the inhibitory signals overwhelm the activating signals^{57, 58}. NKG2D recognises stress- induced ligands such as ULBP1-4 and MICA/B on virus-infected, transformed, or DNA damaged cells, contributing to immunosurveillance^{59, 60}. NKG2E, one of the lesser known NKG2 receptors, can also signal through DAP12 just like NKG2C. However, the complex is typically retained intracellularly at the endoplasmic reticulum⁶¹.

The missing-self hypothesis is intrinsically insufficient to explain NK-cell mediated cytotoxicity without any form of recognition of targets and thus activating signals were predicted in the hypothesis before they were discovered⁶². When a cell is stressed by viral infection or tumorigenic transformation, it can downregulate expression of HLA molecules and upregulate stress-associated ligands as “danger” signal that can be recognised by immune cells^{54, 55, 59}. These changes tip the balance in favour of activating NK cells towards cytotoxicity. Some of these ligands were detected by NKG2D as mentioned earlier^{54, 59}.

NKp30, NKp44 and NKp46 are a group of natural cytotoxicity receptors (NCRs) among the activating receptors expressed by NK cells63, 64, 65, 66. With the exception of NKp44 expressed upon IL-2 cytokine stimulation^{65, 67}, NKp30 is expressed in almost all NK cells and NKp46 is the most reliable marker for NK cells across species. NKp30 and NKp44 have been found to recognise ligands on tumour cells while NKp44 and NKp46 were postulated to recognise viral hemagglutinin leading to cytotoxicity against influenza infected cells^{68, 69}.

Some of the activating receptors perform dual function in the immune synapse. DNAM- 1 is one of them. It is an adhesion molecule that binds to PVR (CD155) and Nectin-2 (CD112), both stress-induced ligands which are overexpressed on tumour cells and CMV infected cells, that produce activating signals in NK cells^{70, 71}. Its expression is also correlated to education and differentiation status of NK cells where educated NK cells are found to express more DNAM-1 against uneducated cells⁷². Furthermore, DNAM-1 is essential for memory formation in murine NK cells during mCMV infection⁷³.

Finally, another important activating receptor on NK cells is CD16. CD16 is a Fc gamma-3 receptor that binds to the Fc region of IgG antibodies on opsonised cells to induce an activating signal through FcRγ and CD3ζ from its intracellular tail⁷⁴. This leads to antibody- dependent cell cytotoxicity (ADCC) upon the opsonised cell⁷⁵. This activation signal through

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CD16 is so strong that it is possible to overcome downstream inhibitory signal generated by KIRs to induce NK activation without co-stimulation from other activating receptors⁷⁵.

1.1.2 NK CELL EDUCATION

Figure 2. NK cell education. MHC class I (in blue and red on the educating cell) are expressed in educating cells to inhibit NK cells in the presence of stimulatory ligands.

The inhibitory signal(s) not only induce tolerance but also establish functionality within NK cells in the form of “education” to respond to a rogue cell that loses MHC I expression. If the NK cell does not express the relevant KIR receptor to detect the MHC I, it cannot generate an inhibitory signal from the encounter and will not be

“educated” and thus remain uneducated and hyporesponsive.

In T and B lymphocytes, their development phase has input a safeguard against autoimmunity through positive selection and negative selection to allow functional yet tolerant lymphocytes proceed forth to their designated purposes respectively. However, for “natural”

cytotoxicity to take place, a similar process should be existent to ensure tolerance with function. Therefore, maturing NK cell should achieve some form of functional capacity that will enable it to not only perform the necessary logic function computation between inhibiting and activating signals but also the relevant response (cytotoxicity or cytokine release) upon activation⁷⁵. Indeed, studies has shown that beta-2-microglobulin (β2m) deficient mice had normal frequencies (albeit hyporesponsive) NK cells despite their inability to express MHC class I on cell surface due to β2m deficiency⁷⁶. Similarly, an appreciably large subpopulation of NK cells in human and mice were found devoid of any expression of KIR or Ly49 respectively and were hyporesponsive just like those in β2m-deficient mice^{77, 78}. This phenomenon of functional calibration by self MHC class I was first demonstrated in mice by Kim et al., who described it as “licensing”, where NK cells achieve functional capabilities upon inhibitory interactions between Ly49 and MHC class I antigen⁷⁹. This functional calibration against the MHC environment enable NK cells to respond robustly to aberrant cells that have downregulated their HLA class I and/or over express stress-associated ligands of activating receptors. Thus, cells that fail to express self-specific inhibitory receptors are not deleted but kept in check by failing to achieve full functional maturation. The term “education” has also been used to describe this process^{80, 81}. To better illustrate the complexity of this process where NK cells achieve functional capacity to perform its logic computation function and act upon it, we will refer it as NK cell education in this thesis^{80, 81}. To summarise Alvin Toffler – Education, in its essence, is a lifelong pursuit to learn, unlearn and re-learn.

In human, NK cell education is achieved through the interaction between inhibitory KIRs and their cognate HLA-A/B/C ligand summarised in figure 2^{78, 80, 81}. As proposed in paper S3, when NK cells encounter an inhibitory interaction through their KIR, they generate

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functional potential through build-up of granules which provides both signalling and cytotoxic potential to respond to a target that has reduced expression of self-HLA antigen⁸². Inhibitory interactions between KIRs and HLA antigens thus educate NK cells to recognise its self HLA and retain the information in their granules which is postulated to retain calcium ions (Ca²⁺) to be used to boost calcium signalling upon activation in addition to producing a more robust degranulation in the immune synapse to achieve a more successful cytotoxicity. Interestingly, the converse occurs with activating 2DS1 where cells expressing 2DS1 are functionally downtuned in donors expressing its cognate ligand HLA-C2⁸³. Such downtuning is stronger in donors who are homozygous for HLA-C2⁸⁴. Furthermore, NK cell education is dynamic as a cell can “unlearn” and “re-learn” according to the MHC environment it is exposed to^{85, 86}. The complexity of the educating interaction based on latest studies has been around cis/trans interactions and transfer into a new MHC environment⁸⁵. Adoptive transfer of uneducated NK cells from MHC deficient mice to MHC sufficient mice leads to gain of functional capacity which is reversed when educated NK cells from a MHC sufficient mice is transferred to a MHC deficient mice^{86, 87}. Another study on humanised 3DL1 in mice model also suggest that KIR-HLA interaction in cis- and trans- configuration are involved in education where trans interaction establish education in NK cells which is then maintained through cis-interaction on the cell⁸⁵. HLA-BW4 presented in trans not only educated NK cells in vivo in 3DL1+

transgenic mice, but NK cells transferred from mice where 3DL1 was absent also became functional upon transfer into these 3DL1+ transgenic mice⁸⁵. The role of cis interaction in NK cell education was also shown when previously educated NK cells’ functional capacity was reduced upon β2m silencing with small hairpin RNA prior to transplant into a host that do not express the cognate ligand⁸⁵.

Despite new information in recent months suggesting a possible mechanistic explanation of NK cell education, the NK cell field remains divided among various fractions with opposing views on NK cell education mechanism⁸². Based on concepts established in 2008, the “arming”, “disarming” and “rheostat” model has been proposed to explain NK cell education and has been dominating the field for the past decade88, 89, 90, 91, 92, 93, 94. The

“arming” model suggest that receptor-MHC class I antigen inhibitory interaction also induces positive signals leading to education of NK cell^{88, 89}. The “disarming” model (illustrated in figure 2) suggest that NK cells that lacked self-MHC class 1 interaction are rendered hyporesponsive from their chronic stimulation encounters with other cells^{90, 91, 92}. The

“rheostat” model seeks to suggests that NK cell education is fine-tuned by the total inhibitory input received where a cell with more inhibitory input during education achieve greater functional capacity^{93, 94}.

A novel idea that is being proposed seeks to explain NK cell education by bringing together all the concepts together towards a central idea termed as “Newtonian” model⁸². The “Newtonian” model is based upon the third law of motion where a force exerted is met with an equal opposing force. Therefore, a NK cell establishes its functional capacity one interaction at a time as it interacts with every cell that it encounters⁴⁹. Inhibitory interactions between KIR and HLA ligand protects NK cells from disarming cell interaction which lead to an opposite positive signal to build up functional capacity in granules shown in figure 2 on the educated cells. Stimulatory interactions between activating receptors and its ligand can also trigger loss of functional capacity through depletion of functional capacity in line with disarming model⁵⁸. The rheostat model then describes the summation of both the strength and number of inhibitory interactions a NK cell receives leading to a diversity in education⁹³. Just as an organism (humans included) receives its education through interactions with its environment, with every attempt, every encounter, every time, throughout its lifespan, so

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does NK cells, learning, unlearning and relearning to fine tune itself functionally to adapt to its ever-changing environment. This is essential for a cell to patrol the organism searching for its target to kill based upon a logic combination of signals from activating and inhibiting receptors. Typically, it will err on the safe side to be more tolerogenic with dominance of inhibitory KIRs and ensuring that only such cells can be fully functional to perform cytotoxicity.

Essentially, NK cell education renders NK cells functional but the lack of education in uneducated NK cells does not eliminate these cells. Rather, it renders them hypofunctional.

Therefore, these uneducated NK cells have the potential to contribute to the human immune response if appropriately recruited and educated later.

1.1.3 NK CELL DIFFERENTIATION

Like other lymphocytes, NK cells developed from CD34+ hematopoietic stem cells which differentiates into common lymphoid progenitor, then commit to NK cell lineage purportedly driven by transcription factors such as inhibitor of DNA binding 2 (ID2) and E4BP4 which inhibits B cells and T cells commitment^{95, 96}. Much of the NK cell differentiation pathway after branching away from B and T cells towards resting NK cells remains a field of intense research with a few speculations along the way, complicated by the discovery of innate lymphoid cells (ILC) which NK cells is just one of the few subtypes within the group⁹⁶. One of the speculations proposed that NK cell precursors do not always remain in the bone marrow to complete its maturation to resting NK cells. NK cell precursors have been found in other lymphoid organs such as liver, lymph nodes and thymus as well as the uterus and mucosal associated lymphoid tissues, suggesting extramedullary maturation is possible^{97, 98, 99}. A recent review by Yu et. al. in 2013 suggested that NK cell development can be divided into 6 stages in both human and mice¹⁰⁰. Stage 1 cells are committed to lymphoid lineage. A population of CD34+CD45RA+CD10+CD117- stage 1 cells in human secondary lymphoid tissues was identified by Freud et al. that retains potential for NK cell, T cell, and dendritic cell (DC) differentiation, supporting the possibility of extramedullary maturation¹⁰¹. Stage 2 was designated to be pre-NK progenitors which is followed by NK progenitors in stage 3 where it has lost the potential to give rise to T cells and dendritic cells. However, these two stages are also complicated with presence of ILC precursors which suggest that these stages are shared lineages among most ILCs. Stage 4 delineate CD56 bright immature NK cells which matures into stage 5 CD56 dim mature NK cells forming the population of resting NK cells. It is worth noting that this lineage remains debatable with many conflicting evidences contesting the linearity between stage 2 and 3, 4 and 5. Nevertheless, it will suffice here to adopt this lineage for its simplicity.

Subsequently, further studies into stage 5 CD56dim NK cell revealed a large heterogeneity within this group of NK cells with a proposed path of differentiation independent from education associated with decline in proliferative capacity, cytokine release, changes in expression of surface molecules involved in activation and homing¹⁰². This begins with CD56bright KIR-NKG2A+ NK cells (stage 4) differentiating into CD56dim NK cells¹⁰². It is found that NKG2A expression is lost as NK cells differentiates¹⁰³. At the same time, NK cells begin to express KIR in a stochastic manner as they differentiate towards terminal differentiation stage which is characterised by expression of CD57^{102, 103}. It is thus reviewed by our group that NK cell differentiation involves a continuous process through cellular interactions to direct NK cells along the differentiation path where there are multiple pathways mediated by education and receptor changes toward terminal differentiation⁸². Therefore, there are vast variations in functional capacity established during differentiation. Apart from the general principles encompassing KIRs, NKG2A and CD57, based upon previous

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publications¹⁰³, the true nature of NK cells functional potential generation and differentiation remains to be elicited. The delineation between subsets are transient and liable to changes by the environment such as cytokines or target encounters. Furthermore, the process of NK cells memory generation has yet to be elicited and remains a topic for further research.

The combination of NKG2A and KIR expressed on NK cells on their differentiation status has been a conundrum to be resolved. NKG2A and KIR provide different schools of education to NK cells¹⁰⁴. NK cells expressing either one of them will receive inhibitory signals that will enable them to respond to cells that has lost the cognate ligand¹⁰⁴. NKG2A, interacting with HLA-E, is postulated to be a more primitive form of NK cell education¹⁰⁵. These NK cells surveyed the body for cells that has lost/reduced HLA-E expression¹⁰⁶. HLA- E typically presents signal peptide of HLA antigens on its peptide binding groove¹⁰⁷. Reduction in HLA antigen expression reduced HLA-E present on the surface of the cell. Most cells normally expressed HLA class I and thus HLA-E would always be present on the cell surface of almost every cell¹⁰⁵. Therefore, NKG2A expressing NK cells would always encounter its cognate ligand and become educated¹⁰⁵. Being among the first inhibitory receptor and a more primitive form of receptor to be expressed in NK cells, expression of NKG2A represent a milestone in NK cell differentiation¹⁰⁵. However, the conundrum arose when we try to determine the origins of NKG2A⁺KIR⁺ and NKG2A^-KIR⁺ NK cells and it is postulated that the latter arise after the former when NK cells lost its expression of NKG2A¹⁰⁶. Thus NKG2A^-KIR⁺ NK cells are postulated to be more differentiated. This does not rule out the possibility that NKG2A^-KIR⁺ NK arose from NK cells that has never expressed NKG2A before¹⁰⁷. The actual mechanism remains unexplored. Unpublished data from our research has revealed an observation that NKG2A can be expressed in NKG2A^-KIR⁺ NK cells after cytokine exposure. Again, the mechanism has not been elicited yet. Based on available published data, our working model is that NKG2A^-KIR⁺ NK cells are more differentiated than NKG2A⁺KIR^-/+ NK cells.

Currently, it has been established that KIR^-NKG2A^-CD57^- NK cells have the least functionality among CD56dim cells¹⁰³. It is still possible that such subset is derived from NK cells that has lost its expression of NKG2A without expressing KIR^{102, 103}. Expression of KIR is postulated to be the next phase of differentiation from KIR-NKG2A+CD57- cells within CD56 dim subset where NK cell education was established^{102, 103}. Expression of NKG2A with an uneducated KIR(s) will also render the NK cell hyporesponsive as compared to a NK cell that express educated KIR(s). The loss of NKG2A after expression of KIR is also proposed to be the next phase of NK cell differentiation¹⁰³. If a NK cells do not express an educated KIR, these uneducated NK cells will become even more hyporesponsive. Functionally, while educated KIR+NKG2A+ NK cells appear to be more responsive; it has been proposed that educated KIR+NKG2A- NK cells are better at cytotoxicity¹⁰⁸. The final phase of differentiation before development of memory will be expression of CD57. Interestingly, expression of CD57 is independent of KIR education and NKG2A expression and remains to be further researched upon for a landmark work to explain the mechanism^{102, 103}.

Finally, terminally differentiated NK cells (CD57+) can differentiate further into

“adaptive” (or memory) NK cells¹⁰². Stage 6 is a novel stage discovered in the 21^st century where the presence of memory NK cells was discovered and confirmed by several researchers in the field11, 12, 102. These NK cells are long lived, persisting longer than 2 weeks, have a highly-differentiated marker phenotype and was found to be excellent killers^{11, 12}. As discussed in the following section, there is an emerging interest in harnessing their unique functional properties in cancer immunotherapy¹⁰⁹.

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1.1.4 Adaptive Natural Killer Cells

Various NK cell-based products have been used to achieve tumor eradication or reduction in cancer patients with cancer, especially leukemia¹¹⁰. Recent efforts have been directed towards developing this specific subpopulation of terminally differentiated NK cells, often referred to as adaptive NK cells for immunotherapy¹¹¹. One form of these adaptive NK cells express NKG2C. They are found at varying frequencies in a fraction of CMV seropositive donors and can be efficiently expanded in vitro with feeder cells overexpressing HLA-E in culture with IL-15 stimulation¹¹². The expansion approaches were reviewed in Paper S1.

Typically, these NK cells exceptionally express only single self-HLA class I specific KIR constituting up to 75% of all NK cells in some healthy donors ¹¹². There are also exceptions with two KIRs, but rarely more. Together, they all have preferential expression of inhibitory KIR specific for self HLA class I molecules112, 113, 114. As shown in paper II, transfer of adaptive NK cells across HLA class I-barriers maximized the effects of a “missing self” response. Apart from expressing NKG2C, these adaptive NK cells do not express NKG2A, even after cytokine stimulation, rendering them an excellent effector cells against tumor cells that overexpress HLA-E, either spontaneously or via immune escape¹¹⁵.

Previously, NK cells were regarded as short-lived innate lymphocytes without developing any memory towards encountered foreign targets. However, this assumption has been refuted with evidence of NK cell repertoire changes after viral infection which engender specific protection against successive infections116, 117, 118, 119, 120. Sun et. al. has shown in mice studies that viral protein m157 recognised by Ly49H⁺ NK cells expanded after CMV infection¹¹⁹. These viral specific NK cells proliferate faster and display stronger effector function upon re-exposure to CMV, resembling CD8⁺memory T cells. Furthermore, the similar NK cell adaptive behavior is also observed in Rhesus Macaques¹³ and human NK cell compartment^{121, 122}. Such CMV-driven expansions of NKG2C+ NK cells have been documented in healthy individuals^{112, 123} and in both solid organ transplantation (SOT)¹²⁴ and hematopoietic stem cell transplantation (HSCT)^{113, 125}. Similar elevation of NKG2C⁺ NK cells in CMV-seropositive individuals have also been observed in other virus infections, including hepatitis C, chikungunya and hantavirus112, 121, 122, 126, 127. It is postulated that these acute or chronic viral infections led to subclinical reactivation of CMV triggering the expansion and long-term persistence of NKG2C⁺ NK cells

Although CMV infection and re-activation remains a significant cause of morbidity and mortality in immunocompromised HSCT recipients¹²⁸, there is epidemiological link between CMV reactivation and relapse protection¹²⁹. This nurtured the idea that emergence of adaptive NK cells early after transplant may contribute to the elimination of leukemic cells.

This concept, while appealing, contradicts Paper II’s observation that adaptive NK cells express self-specific inhibitory KIRs, which effectively abrogate recognition of HLA-matched leukemic blasts. Therefore, it is necessary to study the role of adaptive NK cells in transplantation outcome with respect to the role of HLA mismatch in this context. There is a possibility of the emergence of large populations of highly cytotoxic adaptive NK cells with effector response against the HLA-mismatched targets.

Adaptive NKG2C⁺ NK cells have a differentiated phenotype that can be identified by the following characteristic markers of reduced CD62L, CD7, CD161, CD122, FcεR1g, NKp30, NKp46, siglec-7, siglec-9 expression and elevated CD2, ILT2, CD57 and granzyme B expression¹³⁰.

As mentioned earlier, NKG2C ⁺ adaptive NK cells are just one member of a spectrum of adaptive NK cells. Adaptive expansions are observed independently of NKG2C, in NK cells expressing activating KIR (such as 2DS2, 2DS4)¹¹² or via CD2 as shown in Paper S2. This

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is supported by evidence from a study on patients carrying a homozygous deletion of the NKG2C gene who underwent umbilical cord blood transplantation (UCBT) and developed CMV infection with emergence of activating KIR expressing adaptive NK cells¹³¹. As indicated in Table 1, the natural ligands of some activating KIRs are unknown¹³² but we can speculate from some of the known receptors that they recognize virally encoded ligands in a fashion like Ly49H¹³³.

It is anticipated that adaptive NK cells have an enhanced potential of conventional

“missing self” recognition of partial or total HLA class I loss, secondary to spontaneous loss or induced by T cell- immunotherapy. Thus, such NK cell-based strategies could be a rescue or adjunct therapy for patients undergoing tumor specific CD8 T cells immunotherapy, and checkpoint inhibition.

1.1.5 NK CELLS IN HEALTH AND DISEASE

NK cells are among the first responders to a viral infection contributing to both control of the infection by killing viral infected cells and modulating infection through its interplay with dendritic cells bridging the innate immune system with the adaptive immune system^{134, 135}. NK cell cytotoxicity against viral infection is observed within 3 days of infection as compared to adaptive immune response that requires at least a week after infection to muster¹³⁵. Furthermore, cytokines produced by NK cells are essential to induce further immune response by other immune cells. For example, interferon gamma (IFNγ) is best known to be secreted by activated NK cells in response to stimulation which can activate macrophages to facilitate their respiratory burst and induce upregulation of MHC class I expression for T cell recognition. NK cells are especially essential against herpes virus family infection as shown by patients with NK cell deficiencies. While NK cell deficiencies are not as detrimental as T cell deficiencies, people with only NK cell deficiency are susceptible to severe viral infections and most of them do not survive until adulthood^{136, 137}. Interestingly, NK cells also play a decisive role in providing innate cell protection against Ebola virus; therefore, the role of NK cells in the defence against viral infection cannot be underestimated^{138, 139}. The same applies to cancer immunosurveillance.

The idea of cancer immunosurveillance was proposed in 1909 by Paul Ehrlich who suggested that our immune system can repress occurrence tumours. The formal hypothesis of cancer immunosurveillance was proposed by Sir Macfarlane Burnet and Lewis Thomas in 1957¹⁴⁰. However, the concept did not gain much acceptance until almost a century later when Schreiber and his team was able to show in immunodeficient mice that they are more susceptible to chemically induced tumours^{141, 142}. This tumour is highly immunogenic and can be cleared when transplanted to an immunocompetent host. This has led to an increasing interest in cancer immunotherapy that culminated in many clinical breakthroughs in cancer immunotherapy. Most of the discoveries were made in targeted therapies with antibodies and small molecules, followed by cancer vaccines as well as cellular therapies with T cells and dendritic cells. These efforts have reaped great results in the year 2013. Thus, the year 2013 was named as the year of immunotherapy. Schreiber then proposed in 2002 the concept of immunoediting with the 3 “E” s of immunoediting: Elimination, Equilibrium and Escape¹⁴³. Elimination involves the process of immunosurveillance where stressed aberrant tumour cells were detected and eliminated by the immune cells. The immune system exerts Darwinian selection pressure with phenotype editing of the tumour towards a dynamic equilibrium where the immune system and the aberrant cells are in a state of truce where the tumour does not show significant growth but cannot be eliminated by the immune system144, 145, 146. When the aberrant cells attain sufficient mutations or epigenetic changes to render resistant to the

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immune system’s detection and/or elimination, it has been immunoedited to escape from the immune system.

NK cells’ role in immunosurveillance was illustrated by O’Sullivan et al. in mice¹⁴⁴. When comparing RAG2-/- mice which lacked adaptive immunity against RAG2-/- x [gamma]c-/- mice which also lacks NK cells for their ability to resist methylcholanthrene- induced sarcomagenesis, the latter shown higher incidence of sarcoma¹⁴⁴. This shows that NK cells could control tumours in the absence of adaptive immunity. In humans, the immunosurveillance was illustrated by comparing NK cell cytotoxic capability with risk of cancer ^{147, 148}. So far there is only one prospective study tracing NK cytotoxicity with cancer incidence by Imai et. al¹⁴⁹. Here, 3625 individuals were analysed for peripheral-blood mononuclear cells (PBMC) cytotoxicity against K562 cells (inferred to be mediated by NK cells) and followed for 11 years for cancer incidence and death from all causes¹⁴⁹. Among the 154 cancer cases detected, the study indicated that individuals with medium-high PBMC cytotoxicity was associated with reduced cancer risk while the low cytotoxicity group had increased cancer risk, implying NK cell’s role in cancer immunosurveillance¹⁴⁹. This epidemiological study provided evidence to suggest that NK cells can recognise primary human tumour and eliminate them.

The dearth of evidence on NK cells in immunosurveillance as compared to T cells posit a need for more studies to support the observation made by Imai et.al. to proof that NK cells hold great potential awaiting to be discovered.

1.2CHIMERIC ANTIGEN RECEPTORS (CAR)

Chimeric antigen receptors (CAR) are proteins that are genetically engineered to be expressed on the surface of a cell to alter its function. The typical carrier cell in such cases is a T cell and the protein is typically a fusion protein containing a single-chain variable fragment (scFv) of an immunoglobulin connected to one or more intracellular signalling domains that recognise a specific and unique part of a protein motif (antigen) on the target cell. However, CAR can be used on other cells and does not need to be an immunoglobulin.

There have been examples of NKG2D-CAR engineering of primary NK cells to increase recognition of stressed cells and boost the function of NKG2D¹⁵⁰. Furthermore, there are CARs expressed on NK-92 cell lines for targeting of solid tumours^{151, 152}. Current developments of CAR have increased the variety of hosts available and protein combinations for re-directing the host^{153, 154}. Since then, there are numerous CAR constructs, targets and effector cells that are being used to target various tumours and haematological malignancies in clinical studies around the world154, 155, 156, 157, 158, 159, 160, 161, 162.

Targeting native, unprocessed surface antigens, a conventional (scFv) CAR enables the host effector cell to detect its target independent of protein processing and presentation on MHC antigens. Therefore, they are resistant to downregulation of MHC antigens and problems with peptide presentation that plague T cell receptor (TcR) based therapies for such targets¹⁶³. In the case of a downregulation of MHC class I, a CAR redirected NK cells could experience missing-self with CAR-induced activation; creating possibilities to direct NK cells to new targets and overcoming escape. Furthermore, the use of scFv from immunoglobulin would enable the re-directed cell to target beyond proteins to target non-peptide antigens such as carbohydrates and phospholipids. If a monoclonal antibody can be created against a molecule, we can create a CAR to target it. Similarly, a non-native receptor of the host can be engineered to enable the host to recognise ligands that it naturally is unable to, inducing the desired effects. Also, we can engineer overexpression or improved function of a native receptor on cells with CAR¹⁵⁰. Some examples include re-directing T cells with NCRs,

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overexpressing NKG2D on NK cells¹⁵⁰, expressing an optimised variant of CD16-158V to improve ADCC¹⁶⁴ and CTLA4 in a fusion protein that induces an activating signal on the host¹⁶⁵. The possibilities are limitless.

1.2.1 CAR ENGINEERING

CAR engineering has been around for decades, starting from the first publication by Gross and colleagues in 1989 of a prototype CAR¹⁶⁶. Researchers have since optimised the CAR-design to enhance T cell effector functions and prolong in vivo survival of such redirected T cells. As the groundwork on CAR were based on T cells, CD3ζ was one of the first intracellular subunit used in Ig-fusion receptor protein to study its role in T cell signalling¹⁶⁷. Cross-linking of these fusion proteins (termed T-bodies) lead to calcium flux and T cells activation signalling, laying the basis for development of the first-generation CAR where tumour directed scFv was fused with CD3ζ or FcεR1-γ molecules^{167, 168}. It is subsequently established that CD3ζ subunit is superior over the latter to induce sufficient activating signal that mimic signal 1 generated by the complete CD3 complex¹⁶⁸. Activating via CD3ζ involves tyrosine phosphorylation of the ITAM leading to recruitment of ZAP70, subsequent activation of several cytoplasmic protein kinases in mitogen-activated protein kinase (MAPK) signalling cascade as well as NFAT family of transcription factors^{169, 170}.

The signal 1 generated from this first-generation CAR is liable to anergy and activation induced cell death (AICD) without exogenous co-stimulation of a signal 2 and thus clinical responses were sub-optimal due to lack of persistence after transfer¹⁷¹. Subsequently, CAR engineering was modified to include signalling domains to deliver both signal 1 from CD3ζ and signal 2 from various T cell costimulatory molecules: CD27, CD28, CD134 (Ox-40), CD137 (4-1BB), DAP-10, and inducible T-cell co-stimulator (ICOS)172, 173, 174. The second- generation CARs displayed efficient co-stimulation of T cells without their ligands in vitro with the primary activation signals and showed improved anti-tumour efficacy in vivo in immunodeficient mice models175, 176, 177. This is further supported with clinical efficacy in six lymphoma patients treated with a mixture of first and second generation CD19-CAR-T cells where Savoldo et. al. showed better persistence of second generation CARs¹⁷⁸. Variations of third generation CARs have been made by adding signalling domains of “late” costimulatory molecules from CD134 or CD137 in tandem with CD28 to promote sustained activation and upregulation of anti-apoptotic proteins for better resistance to apoptosis^{172, 179}. In recent months, a robust development in CAR signalling design has led to a huge variety of second-generation CAR with more precise control over its function. These include conditional CAR that requires a small molecule to “switch” on signal 1 and 2, dual CAR where signal 1 and 2 are located on different scFv to allow activation upon presence of both ligands, and a safety CAR that has an inhibitory domain that is co-expressed with standard CAR to allow activation only in the absence of the safety ligand156, 180, 181, 182.

Apart from the intracellular domains, the scFv domain are also actively being developed to improve its specificity, safety and reduce off-target response. These include adding a chemokine receptor, selection marker or a tandem scFv domain to enable dual binding¹⁸². Adding a chemokine receptor will enhance tumour homing and direct the host towards its target by cell trafficking¹⁸². A selection marker or suicide marker will enable physicians to put on emergency brakes upon the cells in times of adverse reaction¹⁸². The tandem domains will enable more precise targeting by requiring both ligand to be present to generate an activation signal. The same applies to dual CAR that also have similar goal as a tandem CAR, ensuring that only the tumour is targeted and leave normal cells alone¹⁸².

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1.2.2 CAR-ENGINEERED T CELLS

CAR engineered T cells came about at the same time as TCR engineered T cells elaborated later. Over time, studies had established many of the current approaches in T cells engineering. The first hurdle is the expansion of T cells to generate sufficient effectors to produce a clinical response. Studies in mice revealed that differentiated effector T cells from long term expansion persisted poorly in vivo and failed to generate comparable anti- tumoral effect as less differentiated central memory T cells^{183, 184}. This was postulated to be due to the latter’s ability to self-renew and differentiate into effector T cells which has lost this ability. Therefore, current approaches for ex vivo expansion of T cells typically last 10-12 days to generate a T cell product that is a mixture of effector memory and central memory T cells179, 185, 186. The importance of the retained ability to regenerate, proliferate, and differentiate is shown in a case study by Kalos et. al. They showed more than one-thousand- fold expansion of CAR redirected T cells in vivo over three weeks from a starting infusion of 1.5x10⁵ cells per kilogramme body weight resulting them constituting 20% of circulating lymphocytes and showing clinical response¹⁸⁷.

There are also several approaches to expand T cells for CAR redirection before or after expansion. One of them is co-culturing T cells with CD3/CD28 antibodies coated on beads¹⁸⁸. This is one of the most common and typical approach used in many centres. Other approaches include agonistic anti-CD3 antibody OKT-3, IL-2, and irradiated allogenic feeder cells¹⁸⁶, and K562-based artificial antigen presenting cells genetically-modified to express a selective antigen with various co-stimulatory ligands, CD64 and membrane bound IL-15^189,

190. Interestingly, this genetically-modified K562 was also found to induce NK cells expansion and is currently used to generate expanded NK cells for clinical studies191, 192, 193, 194.

There are various gene transfer approaches that could be used to transform an effector cell. Most TCR and CAR studies have used optimised gamma-retroviral vectors or lentiviral vectors to transform effector cells178, 195, 196, 197, 198, 199, 200, 201. Retroviral vectors are easy to use with a rich knowledge base and experience from previous in vitro and in vivo studies but are limited by insertional mutagenesis which are now very rare due to the use of dividing cells for transduction²⁰². Lentiviral vectors can also infect non-dividing or minimally activated effectors and carry a larger genetic luggage and are built to prevent replication- competent virus from recombination. An alternative to viral transduction is mRNA transfection where effectors are redirected by mRNA electroporation to express TCR or CAR. The short half-life and transient expression will require multiple infusions to achieve efficacy but offers a safeguard against toxicity and better safety profile. Another non-viral approach would be through DNA integrase/transposon systems such as Sleeping Beauty transposon system which has a more permanent expression of the inserted gene¹³⁵.

1.2.3 CLINICAL APPLICATIONS OF CAR-ENGINEERED T CELLS

Clinical application of CAR T cells has been receiving a lot of attention in public since the success of CAR-T cell therapy in “curing” B cell acute lymphocytic leukaemia (B-ALL) in adult and paediatric patients in the last decade. As of beginning of 2017, there are more than 116 trials on CAR-T cell therapy registered under clinicaltrials.gov, 49 of which were on CD19 CAR-T cell therapy (CART19), the rest covered a wide range of disciplines from carcinoma of brain, breast, liver, gut and renal origins to sarcomas. Many have been completed, evaluating safety and feasibility of adoptively transformed CAR-redirected T cells. Some trials have been initiated to monitor the long-term outcomes of CART19 therapies, a sign of maturity for immunotherapy development. Beginning with leukaemia, CART19 therapies has since been applied to a wide variety of lymphomas. On 30^th August 2017, Kymriah^TM

Engineering NK Cells Towards Next Generation NK Cell Immunotherapy