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Perspective The Journey from Discoveries in Fundamental Immunology to Cancer Immunotherapy Jacques F.A.P. Miller1,* and Michel Sadelain2,* 1The

Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC 3050, Australia Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA *Correspondence: [email protected] (J.F.A.P.M.), [email protected] (M.S.) http://dx.doi.org/10.1016/j.ccell.2015.03.007 2The

Recent advances in cancer immunotherapy have directly built on 50 years of fundamental and technological advances that made checkpoint blockade and T cell engineering possible. In this review, we intend to show that research, not specifically designed to bring relief or cure to any particular disease, can, when creatively exploited, lead to spectacular results in the management of cancer. The discovery of thymus immune function, T cells, and immune surveillance bore the seeds for today’s targeted immune interventions and chimeric antigen receptors. Nanos gigantum humeris insidentes. (Like dwarfs sitting on the shoulders of giants.) —Bernard de Chartres (1120) If I have seen further than others, it is by standing on the shoulders of giants. —Isaac Newton (1676) The immune system is the guardian of our organismal integrity in protecting us from infectious and other foreign invaders, such as grafts and certain tumors. Immunology and oncology thus have a long relationship, in evolutionary terms as well as within the biomedical sciences. The two fields have intersected again and again over the past half-century. We recount here some of these events, with a deliberate focus on T lymphocytes—from their discovery to their genetic engineering and use in cancer immunotherapy. Insightful observations into leukemogenesis in mice were at the root of the discovery of thymopoiesis and lymphocyte subsets. Subsequent observations led to the concept of immune surveillance, with eventual controversies on the role of immunity in tumor prevention and protection that were reconciled in the cancer immunoediting hypothesis. As progress was made in our fundamental understanding of antigen recognition, T cell activation, and T cell costimulation, translational researchers began to exploit the accumulating knowledge for cancer therapy. Tumor-infiltrating T cells were harnessed for adoptive cell therapy in a subset of melanoma patients; cancer vaccines were developed in an attempt to amplify endogenous tumor-specific T cell responses. Two of the most recent, most exciting therapeutic developments rest on the manipulation of costimulatory pathways governing T cell function. One, based on monoclonal antibody technology, enables the release of tumor-infiltrating T cells from inhibition mediated by costimulatory receptors such as CTLA-4 and PD-1, through either checkpoint blockade or depletion of regulatory T cells. The other, based on gene transfer technology, enables to repurpose patient’s T cells, targeting them to tumor antigens and augmenting their functional properties to overcome barriers erected by tumor cells and their microenvironment. These two forms of cancer immu-

notherapy were recognized by the Science magazine as the ‘‘breakthrough of the year’’ in 2013 (Couzin-Frankel, 2013) and by the US Food and Drug Administration, which approved the anti-CTLA-4 monoclonal antibody Ipilimumab in 2011 and granted breakthrough status to the CD19-specific chimeric antigen receptors (CARs) utilized at the University of Pennsylvania and Memorial Sloan Kettering Cancer Center for the treatment of pediatric and adult acute lymphoblastic leukemia in 2014. In this review, we aim to expose how basic discoveries in immunology led to these promising advances in cancer therapy. Leukemogenesis, the Thymus and Immunological Ontogeny Prior to 1960, the thymus was thought to be a vestigial organ that had become redundant during evolution and was just a graveyard for dying lymphocytes. Even though recirculating small lymphocytes had been found by Gowans (Gowans et al., 1962) to be immunocompetent cells able to initiate either cellular or humoral immune responses, thymus lymphocytes were deemed immunoincompetent since they did not recirculate, nor could they transfer immune responses to appropriate recipients. Furthermore, thymectomy, which had always been performed in adult animals, had no untoward effects on immune capacity. In 1959–1961, however, results obtained in a mouse model of lymphocytic leukemia induced by the Gross leukemia virus led to experiments using neonatally thymectomized mice. To obtain a high incidence of leukemia in mouse strains that were not highly prone to develop this malignancy, the virus had to be given at birth, and not later (Gross, 1951). Thymectomy of virus-inoculated mice at 1 month of age prevented the disease (Miller, 1959a), and grafting a neonatal thymus as late as 6 months after adult thymectomy restored the potential for leukemia development (Miller, 1959b). Clearly, the virus must have remained latent, and indeed, it could be recovered from the non-leukemic tissues of thymectomized mice not grafted with thymus tissue (Miller, 1960). It was surmised that to induce a high percentage of leukemia in low-leukemic strains of mice, the virus had to be given neonatally because it needed to multiply in some cells present only in a newborn thymus. To test this hypothesis, the virus was given Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc. 1

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Perspective at birth but immediately after neonatal thymectomy, based on the prediction that thymus grafting performed later would not restore leukemogenesis. The neonatally thymectomized mice fared well until some weeks after weaning, when many became sick, wasted, and died. This had never been seen by anyone who had thymectomized adult mice (Miller, 1961a). Post-mortem examination revealed lesions in the liver suggestive of mouse hepatitis virus infection and marked diminution of lymphocytes in the blood and in the lymphoid tissues (Miller, 1961b, 1962a). As lymphocytes were known to be involved in graft rejection and other immune responses, the neonatally thymectomized mice were tested for immunocompetence by grafting them with foreign skin before they had begun to show signs of wasting disease. Remarkably, they failed to reject foreign skin grafts, even when donors and recipients differed at the major histocompatibility locus, H-2 (Miller, 1961b, 1962a). The neonatally thymectomized mice also lacked the ability to produce a normal antibody response to certain antigens, such as Salmonella typhi H antigen (Miller, 1962a) and sheep erythrocytes (Miller et al., 1965). Thymus grafting restored immune potential, and grafting of a foreign thymus induced specific tolerance to skin from the donor of the thymus graft (Miller, 1962a). This suggested that the thymus may be the seat where tolerance is learned: ‘‘Antigenic material might make contact with certain cell types differentiating in the thymus and in some way prevent these cells from maturing to a stage when they would be capable of reacting immunologically’’ (Miller, 1962a). This clear prediction of negative selection was proven some years later by using the so-called super antigens and transgenic mice, and both positive and negative selection of thymocytes were then worked out (Bevan, 1977; Kappler et al., 1987). The major events in thymus cell differentiation have been summarized in a recent Timeline review (Miller, 2011). It seemed important to determine whether the thymus exerted its influence by seeding cells into the rest of the lymphoid system. Since no cell-surface markers had at that time been found to identify cells from different locations, use was made of the T6 strain of mice, the cells of which could be identified at metaphase by the presence of two minute chromosomes. Neonatally thymectomized F1 hybrid mice, in which one parent was T6, were grafted with thymus from the other parental strain and immunized with skin from various donors. An analysis of the chromosome constitution of the cells in metaphase in the spleen showed that 15% to 20% had originated from the thymus graft (Miller, 1962a). This suggested that the thymus did produce cells capable of migrating to the periphery and that presumably such recent thymus emigrants would have just matured before leaving or would mature in the peripheral lymphocyte pool to become fully competent lymphocytes. Although adult thymectomy had no effect on immunological capacity, it could conceivably have caused a problem in mice whose lymphoid system had been destroyed in some way, as for example by total-body irradiation. This proved to be the case in partially irradiated mice (Miller, 1962c) and in lethally irradiated mice protected with bone marrow (Cross et al., 1964). The use of adult thymectomized, heavily irradiated mice protected with bone marrow (ATxBM mice) proved invaluable for further elucidation of immune functions. 2 Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc.

All of these results were initially regarded with some skepticism. Vague criticisms abounded, such as the one stating that mice must be unusual and that the results obtained would never be seen in humans. What needed to be checked, however, was that because the mice used had been raised in converted horse stables, the added trauma of neonatal thymectomy made them highly susceptible to infections. This criticism was soon quashed when it was shown that mice reared in a germfree facility, when thymectomized at birth did not develop wasting disease and yet were unable to reject skin grafts, even those that differed at the H-2 locus (McIntire et al., 1964). In 1968, the nude mouse was discovered (Rygaard, 1973), and immunologists no longer had doubts about the immunological function of the thymus. In children with thymus aplasia, as in di George syndrome, thymus transplantation has reversed immunodeficiency (August et al., 1970). The problem lies in donor availability and tissue compatibility. However, the recent spectacular success in creating a functional thymus organ by enforcing Foxn1 expression to reprogram mouse embryonic fibroblasts into fetal thymic epithelium (Bredenkamp et al., 2014) constitutes a first promising step in the provision of appropriate thymus tissue. The Identification of T and B Cells in Mice As stated before, Gowans had clearly shown that recirculating small lymphocytes could respond both by a cellular immune response (as in skin graft rejection) and by producing antibody (Gowans et al., 1962). He considered that the same cell could take part in either, depending on the antigenic stimulus. As neonatally thymectomized mice were deficient in both cellular and at least some humoral responses, it was urgent to show that they had markedly reduced numbers of recirculating lymphocytes, not just blood lymphocytes. This was performed after cannulating the thoracic duct of neonatally thymectomized and ATxBM mice and collecting the lymph over a 24- to 48-hr period (Miller et al., 1967; Miller and Mitchell, 1967). The conclusion was made that most thoracic duct lymphocytes (TDLs) in mice were thymus derived, and this was actually proven in subsequent experiments (Miller and Sprent, 1971). But were the same cells involved in antibody production and skin graft rejection? Two experimental systems suggested that this might not be the case. Claman (Claman et al., 1966) showed that irradiated mice given syngeneic bone marrow cells and syngeneic thymus cells could produce more antibody than when given either cell source alone. As no antibody markers were available at the time, the origin of the antibody-forming cells could not be easily identified. The situation was different for chickens in Burnet’s laboratory, where impairment of bursa function by testosterone injection caused the birds not to produce antibody, whereas thymus atrophy in sick birds prevented graft rejection (Warner et al., 1962). Because mammals do not have a bursa, Burnet (Burnet, 1962) surmised, ‘‘In mammals it is highly probable that the thymus also carries out the function of the bursa of Fabricius in the chicken, which is to feed into the body the cells whose descendants will produce antibody.’’ In 1967 and 1968, Miller and Mitchell (Miller and Mitchell, 1967, 1968, 1969), reconstituted neonatally thymectomized and adult thymectomized, irradiated CBA strain mice with CBA bone marrow and (CBAXC57BL)F1 TDLs and challenged them with sheep erythrocytes. The ATxBM mice in those experiments

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Figure 1. The Origins of T Cells and B Cells Percent reduction of antibody-forming cells per spleen of adult thymectomized and heavily irradiated CBA protected with CBA bone marrow (ATxXBM), given (CBAXC57BL)F1 thoracic duct lymphocytes, and challenged with sheep erythrocytes. To the incubation mixture was added normal mouse serum (NMS; slightly toxic), C57BL anti-CBA serum (aCBA), or CBA anti-C57BL serum (aC57BL) as shown. The number of mice providing spleens in each group was three to six. Adapted from Mitchell and Miller (1968). These experiments demonstrated the existence of thymus-derived cells (later known as T cells) that were not antibody formers but were essential to allow cells derived from the bone marrow (later known as B cells) to produce antibody to certain antigens (later known as thymus-dependent antigens).

were injected with immunocompetent TDLs, and antibody-forming cells were produced that lysed sheep erythrocytes. This was shown by plaques when sheep red cells and spleen cells were layered onto agar plates. Now the time was appropriate to determine whether the TDLs gave rise to antibody-forming cells. It was done by simply adding to the plates antiserum against CBA strain antigens made by immunizing C57BL mice with CBA tissues or antiserum against C57BL made in CBA mice. The latter would be expected to kill any antibody-forming cells if they were derived from the (CBAXC57BL)F1 TDL and hence from the thymus-derived cells (Figure 1). The results were spectacular, showing beyond any doubt that the antibody-forming cells did not originate from the TDLs, but rather from the bone marrow (Mitchell and Miller, 1968). It proved that lymphocytes could be subdivided into two major groups: thymus-derived cells (later known as T cells) were not antibody formers but were essential to allow cells derived from the bone marrow (later known as B cells) to produce antibody in response to certain antigens (later known as thymus-dependent antigens). They were thus helper cells collaborating with other cell types, derived from the bone marrow, to enable these to produce antibody. The murine equivalent of the bursa was thus the bone marrow. The existence of two major lymphocyte subsets was first regarded with some skepticism. At a meeting in Brooke Lodge (Augusta, Michigan) held in 1968, Good was ‘‘concerned at separating thymus-derived from marrow-derived cells’’ since the former ‘‘are in fact marrow derived-cells.’’ He also claimed to ‘‘have evidence that in the rabbit it (the bursa equivalent) resides in the ilial lymphoid tissue and in the lymphoid tissue of the appendix’’ (Good, 1969). At the same meeting, Gowans (Gowans, 1969), who had proven that recirculating small lymphocytes were immunocompetent, stated, ‘‘Had it not been for

Dr Miller’s experiments I would have assumed that a single variety of small lymphocyte was involved in each of our experiments. If we have two cell lines that are collaborating, then we have specificity residing in two cell lines, one thymus-derived and the other marrow-derived. The problem is to bring these two specific cell lines together. Does this necessity for the two cells to find one another raise problems? It seems an inefficient mechanism if it rests only on chance contacts.’’ The then Professor of Immunology at the National University in Canberra, Australia, offered a less diplomatic critique and simply likened B and T cells to the first and last letter of the word ‘‘bullshit.’’ The above somewhat simple experiments, performed without the use of modern technologies such as gene targeting, cluster of differentiation (CD) antigens, monoclonal antibody, and flow cytometry, changed the course of immunology. Thus, the existence of T and B cells required a reinvestigation of numerous immunological phenomena in terms of the role played by the two distinct cell types, including the carrier effect, immunological tolerance, immunological memory, immunodeficiency, autoimmunity, and genetically determined immune responsiveness. An avalanche of work soon followed as investigations were focused on defining and investigating further lymphocyte subsets and their function. It was clear that the antigen recognition receptor on B lymphocytes was immunoglobulin (Ig), but controversy raged concerning the nature of the T cell receptor (TCR) for antigen. Some argued strongly in favor of the existence of a different type of Ig molecule (IgT) (Marchalonis et al., 1972). Others, however, showed that Ig on T cells had originated from B cells and bound to the T cell Fc receptor (Hudson et al., 1974) and that no RNA molecules bearing Ig VH nucleotide sequences could be detected in T cells (Kemp et al., 1982). The discovery of monoclonal antibodies (Ko¨hler and Milstein, 1975) was finally instrumental in elucidating the TCR structure, and its coding sequences were soon cloned independently in mouse and man (Hedrick et al., 1984; Yoshikai et al., 1984). The TCR chains (a and b, or in some cases g and d) are highly homologous to Ig and are associated on the T cell membrane with CD3, a complex of three polypeptides, g, d,and ε, and with a disulphide-linked z-z homodimer or z-h heterodimer (Gold et al., 1986). These complexes not only regulate the assembly and expression of the TCR, but are also responsible for trans-membrane transduction of signals after antigen occupation (Oettgen et al., 1985). The demonstration that the TCR was sufficient to direct antigen specificity in  et al., 1986) would set the stage for transgenic mice (Dembic TCR-based T cell engineering two decades later. Since T and B cells utilize different molecules as antigen-specific receptors, do they perceive antigen in different ways? This is indeed the case: whereas B cells can bind soluble antigen, T cells generally recognize antigen only if displayed on the surfaces of antigen-presenting cells (APCs), such as dendritic cells (Steinman and Cohn, 1973) or virus-infected cells. Cytotoxic T cells isolated from mice recovering from a virus infection were tested for their capacity to kill virus-infected target cells in vitro. Killing was observed but only if the target cells had the same major histocompatibility complex (MHC) haplotype as the mice from which the T cells were obtained (Zinkernagel and Doherty, 1974). This suggested that there might be an association between a virus product and MHC molecules at the cell Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc. 3

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Perspective surface and that the specificity of the TCR was directed to both MHC molecules and virus-encoded products. The phenomenon became known as MHC restriction and the MHC molecules involved as restriction elements. T cells in fact recognize relatively short peptide fragments (Townsend et al., 1986) that become wedged in the jaws of the MHC molecules. How T and B cells perceive antigen is highly relevant for tumor immunity and the escape of immunogenic tumor cells from any host immune response. Loss of cell-surface antigen or MHC molecules from genetically unstable tumor variants would of course prejudice or prevent the response of any specific B and T cells. CARs would later capitalize on the respective advantages of both targeting modalities—and Ig VH genes would make their way into T cells, after all (see below). Further T cell subsets and their function were soon identified: CD4 and CD8 T cells (Kisielow et al., 1975; Cantor and Boyse, 1975), natural killer T (NKT) cells (Makino et al., 1995; Godfrey et al., 2004), and CD4+CD25+FoxP3+ regulatory T (Treg) cells (Sakaguchi et al., 1985). Self-tolerance, as mentioned above, is mostly achieved intra-thymically by negative selection of self-reactive thymocytes. Since, however, some self-reactive T cells (e.g., those with low affinity to self-antigens) escape to the periphery, a peripheral tolerance mechanism exists, enabling Treg cells to prevent autoimmunity (reviewed in Josefowicz et al., 2012). Treg cells are either produced in the thymus during T cell development (natural Treg [nTreg] cells) or induced from naive CD4+ T cells in the periphery (so-called induced Treg [iTreg] cells) and are marked by expression of the transcription factor Foxp3 (Sakaguchi and Powrie, 2007). These cells soak up interleukin-2 (IL-2) (Smith, 1989) through their high-affinity trimeric IL-2 receptor, thus depriving other T cells of this growth factor, but the exact mechanism by which, when recruited into the tumor environment, they prevent the activity of other immune cells capable of destroying the tumor is not clear. It may involve the production of inhibitory cytokines such as transforming growth factor (TGFb) (Powrie et al., 1996), IL-10 (Asseman et al., 1999), and IL-35 (Collison et al., 2007). It is not the aim of this review to describe in further detail each of the key advances that have led to our current knowledge of the function of T and B cells, since this has been extensively covered recently in a Timeline review (Miller, 2011). It is noteworthy that several genes that govern T cell development or function, e.g., those encoding the IL receptor common g chain, the Wiskott-Aldrich syndrome protein, and others, are now being developed as genetic therapies for the treatment of severe immunodeficiencies (Fischer et al., 2013) From Immune Surveillance to Cancer Vaccines and Adoptive T Cell Therapy The finding that neonatally thymectomized mice were more susceptible than normal mice to the cancer-producing activities of strong carcinogens (e.g., 3,4-benzopyrene) (Miller et al., 1963) and polyoma virus (Miller et al., 1964), (Miller, 1967) provided some experimental support for Paul Ehrlich’s concept of immunological surveillance (Ehrlich and Himmelweit, 1957): ‘‘I am convinced that during development and growth malignant cells arise extremely frequently, but in the majority of people they remain latent due to the protective action of the host. I am also convinced that this natural immunity is not due to the presence 4 Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc.

of antimicrobial bodies but is determined purely by cellular factors. These may be weakened in older age groups where cancer is more prevalent.’’ Burnet echoed this view in his paper on tumor immunosurveillance (Burnet, 1971). A large amount of work has been done by many to determine the importance of immunocompetent lymphocytes in guarding against the emergence of neoplastic clones of cells. There is no doubt that a deficiency of such cells is associated with an increased incidence of those neoplasms that happen to be strongly immunogenic and to be produced by strong carcinogens as cited above (Miller et al., 1963). However, extensive studies performed using athymic nude mice failed to provide any evidence of an increased susceptibility to the development of spontaneous tumors (Rygaard and Povlsen, 1976). On the other hand, RAG-deficient mice (lacking T, B, and NKT cells) do spontaneously develop gastrointestinal epithelial malignancies by the age of 18 months (Shankaran et al., 2001); immunocompromised pathogen-free severe combined immunodeficiency (SCID) mice have a high incidence of spontaneous thymic lymphomas, and about 2% of retired breeders developed a variety of non-thymic tumors (Huang et al., 2011). It is well established that immunosuppressed humans, e.g., following therapy for transplantation, have an increased incidence of tumors such as skin or cervical carcinomas and lymphomas (Penn and Starzl, 1973). A key question is whether this is the result of failed immunosurveillance or of increased susceptibility to infection by potentially oncogenic viruses (human papillomavirus, hepatitis C virus, human immunodeficiency virus, and Epstein-Barr virus [EBV]). Many of the antigens expressed by spontaneous tumors are expected to be self-antigens, and hence thymus-derived lymphocytes responding to these antigens would have been deleted in the thymus. Low-affinity cells specific for these self-antigens may, however, have escaped deletion and migrated to the periphery, where they may be held in check by the growing tumors as described below. Some spontaneous tumors have a high frequency of mutations that may therefore generate de novo antigens (e.g., BCR-Abl, mutated forms of p53) that will not have been present in the thymus and may thus appear foreign to the immune system (Coulie et al., 2014; Gubin et al., 2014). In these cases, therefore, both T cells and antibodies directed to tumor antigens should be able to eliminate the tumor. It is, however, not clear how likely it is that a point mutation in a single oncogene would give rise to an antigenic peptide. In some instances, pathogen-elicited thymus-derived cells that cross-react with neoantigens acquired through somatic mutation may contribute to anti-tumor responses (Snyder et al., 2014). The bulk of the observations on immune surveillance have in fact shown that T cells and antibodies could recognize and eliminate tumors (Boon et al., 1994). How tumors spontaneously generate tumor immunity has led to the cancer immunoediting hypothesis (Schreiber et al., 2011). This consists of three sequential phases: elimination, equilibrium, and escape. In the first phase, tumors developing before being clinically apparent are destroyed and eliminated by both innate and adaptive immune mechanisms (e.g., Shankaran et al., 2001). If variants arise and fail to be eliminated, cells of the adaptive immune system may restrain their growth during the lifetime of their host and a state of equilibrium is reached. Constant immune selective

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Cancer Cell

Perspective pressure of genetically unstable tumors may, however, lead to variants that can no longer be recognized by effector T cells because they have downregulated antigen or MHC, secreted immunosuppressive cytokines such as TGFb or IL-10, or recruited Treg cells in their environment. They then enter the escape phase and become clinically apparent. The administration of a cancer vaccine, such as GVAX (Dranoff et al., 1993), has been used in the management of non- or weakly immunogenic cancers. GVAX expresses tumor antigens in the context of two important adjuvant activators, granulocytemacrophage colony-stimulating factor (GM-CSF) and pathogen associated molecular patterns (PAMPs), that promote the activation of T cells with low affinity for self-antigens expressed by tumors. This vaccine, together with the DNA-damage-inducing chemotherapeutic drug cyclophosphamide that preferentially targets Treg cells in a mixed cell population (Ghiringhelli et al., 2007), with further assistance from the dual cocktail of antiCTLA-4 plus anti-PD-1 (or anti-PD-L1) antibodies, discussed below, may be successful in the treatment of certain tumors— if potent, truly tumor-specific T cell responses are indeed elicited. This goal may be harder to reach in tumors with a low rate of mutations (Alexandrov et al., 2013). The focus on T cell responses inspired yet another approach in cancer immunotherapy, pioneered by Rosenberg and colleagues, focusing directly on the patient’s own tumor-infiltrating lymphocytes (TILs) when those exist and are accessible. In TIL therapy, T cells retrieved from surgical melanoma specimens are cultured under activating conditions and reinfused with IL-2 (Rosenberg et al., 1986) after host preconditioning (Gattinoni et al., 2006). This approach is, however, not applicable to all melanoma patients nor many other cancers. In EBV-associated lymphoproliferative disease, for which EBV-transformed B cells uniquely serve as effective APCs for tumor antigens of viral origin, tumor-specific T cell lines have been successfully generated from peripheral blood of healthy donors and used to treat immunocompromised recipients (Papadopoulos et al., 1994; Heslop et al., 1996). Artificial APC systems substituting for EBV-transformed B cells or dendritic cells (Kim et al., 2004) have since been developed to facilitate the selection and/or expansion of therapeutic T cells. From T Cell Activation Biology to Checkpoint Blockade and T Cell Engineering Most spontaneous tumors express self-antigens and present peptides derived from them embedded in MHC proteins on their cell surface. T cells with high-affinity TCRs for these would have been deleted by negative selection in the thymus. In melanomas, which are known to undergo a high mutation rate (Berger et al., 2012), some mutated peptides are displayed as altered selfantigens on the cell surface, where they can be recognized by cytotoxic CD8 T cells. So why are these immunogenic tumors not spontaneously killed by activated CD8 cells? Extensive studies of the molecules and processes involved in T cell activation have shed light on this conundrum and yielded levers to enable or restore T cell function. T cell activation initially requires two signals, one from the recognition of antigen-derived peptides displayed in the context of MHC molecules on the surface of APCs and one from the interaction of the T cell costimulatory molecule CD28 with its

ligands CD80 (B7-1) and CD86 (B7-2) on the APC (Lafferty and Cunningham, 1975; Harding et al., 1992). Additional costimulatory receptors support T cell expansion and the maintenance of T cell memory (Croft, 2003). Both activating and costimulatory molecules would eventually be turned into powerful anti-cancer agents, via very different therapeutic strategies, one based on monoclonal antibody technology and the other on genetic engineering. Reversing Tumor-Protective Immune Inhibition Under normal circumstances, T cell responses are regulated by inhibitory checkpoints, mediated in part by CTLA-4 (CD152), PD-1 (CD279), and BTLA (CD272), to prevent unrestrained multiplication, collateral damage by cytotoxic effector molecules, and even autoimmunity. CTLA-4 competes with CD28 for the ligands CD80 and CD86 and acts as a signal dampener during the early stages of activation of naive and memory T cells, in fact 24–48 hr after antigen presentation (Krummel and Allison, 2011). It has been shown to act as an effector molecule that inhibits CD28 costimulation by the cell-extrinsic depletion of its ligands (Qureshi et al., 2011). CTLA-4 is expressed on T cells, and its main physiological function is to down-modulate CD4 T helper cell activity and increase CD4+CD25+FoxP3+ Treg-cell-mediated immunosuppression (reviewed by Pardoll, 2012). A deficiency or mutation in CTLA-4 in humans has contributed to the development of autoimmune diseases including autoimmune hypothyroidism, type I diabetes, systemic lupus erythematosus, and celiac disease (reviewed by Watanabe and Nakajima, 2012). Recently, a heterozygous nonsense mutation in exon 1 of CTLA4 has been identified in one of the commoner subgroups of common variable immunodeficiency syndromes (CVIDs) characterized by hypogammaglobulinemia, recurrent infections, and multiple autoimmune manifestations (Schubert et al., 2014). PD-1 is expressed following T cell activation and during chronic antigen-induced T cell stimulation, and it limits the activity of T cells that have already been activated (Keir et al., 2008). Unlike CTLA-4, PD-1 is expressed not only on all T cells subsets but also on activated B cells and NK cells. It exerts its main function during the inflammatory response associated with infection. Chronic antigen exposure, as occurs in certain viral infections, can induce exhaustion of cognate antigen-specific activated T cells. Furthermore, signals from inflammatory tissues induce the expression of the PD-1 ligands, PD-L1 (B7-H1) or PD-L2 (B7-DC), that downregulate T cell activity, thereby limiting collateral damage and even autoimmunity. As with CTLA-4, mutations in PD-1 have also been associated with autoimmunity in humans (Watanabe and Nakajima, 2012). Theoretically, therefore, antagonists directed to these immune inhibitory checkpoints should enable T cells to proliferate and respond more vigorously and would be beneficial in controlling virus infections and suppressing tumor growth (Figure 2). Agonists of CTLA-4 or PD-1, on the other hand, may prevent undesirable immune responses as occur in autoimmunity, allergy, and transplant rejection (Okazaki and Honjo, 2007). In many cases, cancer cells express the ligand PD-L1 on their surface, which may restrict tumor-reactive T cell responses by engaging the inhibitory receptor PD-1. Furthermore, PD-L1 on tumor cells and the surrounding stromal cells is upregulated by g-interferon Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc. 5

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Figure 2. Antibody-Mediated Targeting of Negative Regulators of T Cell Responses Anti-CTLA4 monoclonal antibodies can alleviate CTLA4-mediated abrogation of CD28 signaling in T cells and deplete intra-tumoral regulatory T cells (Treg cells), which act exogenously to suppress anti-tumor effector T cells. PD-1/PD-L1 blockade interrupts the PD-L1 tumor adaptive response, which may be initiated by T-cell-derived g-interferon secretion, inducing PD-L1 expression by the tumor cell, and the induction of PD-1 expression by the activated T cells.

secreted by the very activated T cells that have infiltrated the tumor environment (Iwai et al., 2002). Treg cells, which specifically suppress activated T cells during a normal immune response to limit collateral damage and autoimmunity, are recruited to the tumor environment and act as another dampener that limits the activity of tumor-infiltrating cytotoxic T cells (Curiel, 2007). Treg cells express high levels of immune checkpoint receptors, including CTLA-4 and PD-1, wherein these inhibitors of effector T cells paradoxically enhance Treg cell activity. These receptors may thus serve as targets for antibody-mediated Treg cell depletion (Simpson et al., 2013) (Figure 2). A deficiency of CTLA-4 in Treg cells in mice results in the spontaneous development of systemic lymphoproliferation, fatal T-cell-mediated autoimmune disease, and hyperproduction of IgE, and the loss of CTLA-4 also potentiates tumor immunity (Wing et al., 2008). Furthermore, the immune-cell-expressed ligand semaphorin4a (Sema4a) and the Treg-cell-expressed receptor neuropilin-1 (Nrp1) interact to potentiate Treg cell function and survival. Nrp1 is required by Treg cells to limit anti-tumor immune responses, suggesting that the Nrp1-Sema4a pathway may be a potential target to limit Treg-cell-mediated tumor-induced tolerance (Delgoffe et al., 2013). Inhibitory myeloid cells also accumulate in certain tumor types and may negatively impact on anti-tumor T cell responses (reviewed in Ostrand-Rosenberg, 2010). Monoclonal antibody technology (Ko¨hler and Milstein, 1975) has enabled a range of immunotherapies targeting cell-surface molecules. What has recently been achieved with clinical success in the case of some tumors, in particular malignant metastatic melanoma, is the use of blocking monoclonal antibodies directed against CTLA-4 and PD-1 or PD-L1 (Figure 2). These allow the specific tumor infiltrating T cells to undergo unre6 Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc.

stricted proliferation, thereby enhancing their capacity to kill their tumor targets. The blockade of CTLA-4 using monoclonal antibodies, as first shown by Allison (Leach et al., 1996), restored tumor immunity in mice bearing immunogenic tumors. There are two anti-CTLA-4 antibodies in use against metastatic melanoma: ipilimumab and tremelimumab. There are eight anti-PD-1/PD-L1 antibodies in clinical trials: nivolumab, pembrolizumab /MK3475, pidilizumab, and AMP-224, targeting PD-1; and BMS935559, MEDI4736, MPDL3280A, and MSB0010718C, targeting PD-L1. In addition, inhibitors against other immune checkpoint regulators expressed on the cell surface, such as TIM3, LAG3, and VISTA, and agonist antibodies acting on T cell costimulatory receptors, such as OX40 and CD137, are being developed for cancer immunotherapy (European Society for Medical Oncology, 2014). The dual cocktail of ant-CTLA-4 and anti-PD1 has been most effective in treating some melanoma patients (Curran et al., 2010). Monoclonal antibodies targeting CCR4, a chemokine receptor that is highly expressed on Treg cells, and PI3 kinase inhibitors that preferentially block Treg cell activity, may soon complement the T cell activation checkpoint blockade modulators (e.g., antibodies against CTLA4 or PDL1) in immunotherapy of cancer (Sugiyama et al., 2013; Abu-Eid et al., 2014). The Promise of T Cell Engineering The advent of gene transfer technologies, in particular those enabling the transduction of human T lymphocytes using gibbon ape leukemia virus envelope-pseudotyped g-retroviral vectors (Mavilio et al., 1994; Bunnell et al., 1995; Gallardo et al., 1997), created new opportunities for immune intervention based on T cell engineering (Sadelain et al., 2003). Patients’ T cells, easily accessible in peripheral blood, can be genetically instructed to target tumors by transduction of receptors for antigen, utilizing either the physiological TCR (Ho et al., 2003; Stone and Kranz, 2013) or synthetic receptors now known as CARs (Sadelain et al., 2009). Both approaches have shown clinical successes, particularly in melanoma (Robbins et al., 2011), targeting NYESO1 (Chen et al., 1997), and in acute lymphoblastic leukemia, (Brentjens et al., 2013; Grupp et al., 2013; Davila et al., 2014; Lee et al., 2015; Maude et al., 2014), targeting CD19 (Brentjens et al., 2003). CARs are artificial, composite receptors for antigen that integrate principles of B cell and T cell antigen recognition (Figure 3A). They are particularly attractive in that they elude human leucocyte antigen (HLA) restriction and are thus applicable to all patients irrespective of their HLA haplotypes, unlike TCRs. CARs may also overcome HLA downregulation by tumors, which deprives T cells of a ligand for their endogenous TCR (Seliger, 2008). The critical function of CARs is, however, not to merely target the T cells to a tumor antigen, but to enhance T cell function. Thus, effective CARs further integrate principles of T cell costimulation (Figures 3B and 3C) and provide a broad spectrum of functional enhancements acquired by directly soliciting selected costimulatory pathways (Sadelain et al., 2013; Jensen and Riddell, 2015). The CARs that have recently shown impressive clinical outcomes in patients with B cell malignancies are second-generation CARs, so named to highlight their fundamental difference with the CD3z chain fusion receptors that preceded them

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Perspective Figure 3. Principles of T Cell Engineering and CAR Design (A) Integration of B cell and T cell antigen recognition principles in the design of CARs. The heavy and light chain chains, which are components of the B cell receptor and Igs, are fused to the T-cell-activating z chain of the TCR-associated CD3 complex to generate non-MHC restricted, activating receptors capable of redirecting T cell antigen recognition and cytotoxicity. (B and C) Integration of T cell activation and costimulation principles in dual signaling CARs designed to enhance T cell function and persistence in addition to retargeting T cell specificity. In (B), the physiological abTCR associated with the CD3 signaling complex is flanked by the CD28 costimulatory receptor. (C) shows a prototypic second-generation CAR, which comprises three canonical components: an scFv for antigen recognition, the cytoplasmic domain of the CD3z chain for T cell activation, and a costimulatory domain to enhance T cell function and persistence (here CD28, as described in Maher et al. [2002], and currently utilized in ALL patients as reported in Brentjens et al. [2013] and Davila et al. [2014]). Unlike the abTCR/CD3 complex, which comprises g, d, ε, and z signaling chains and is modulated by a multitude of costimulatory receptors, CARs possess in a single molecule the ability to trigger and modulate antigen-specific T cell functions.

(Sadelain et al., 2009). These molecules evolved in three critical stages. When the z chain of the CD3 complex was independently cloned by the Weiss, Seed, and Klausner groups, the newly discovered T-cell-specific chain, which lacks an extracellular domain (Figure 3B), was fused to CD8, CD4, or CD25 (Irving and Weiss, 1991; Letourneur and Klausner, 1991; Romeo and Seed, 1991) to enable antibody-mediated cross-linking of the z chain fusion receptor. These studies, conducted in leukemic T cells, revealed the activating function of the z chain. The addition of an scFv derived from an Ig, first reported by Eshhar and colleagues (Eshhar et al., 1993; Brocker et al., 1993), diversified the binding capacity of these fusion receptors and redirected the specificity of transfected cytotoxic T cell hybridomas (Eshhar et al., 1996). Having established methods for the transduction of human peripheral blood T cells (Gallardo et al., 1997), we could for the first time test the functional features of z-chain-based CARs in clinically relevant cell types. We found z-chain-based

CARs to be unable to command T cell expansion upon repeated exposure to antigen (Gong et al., 1999), consistent with the two-signal model of T cell activation (Lafferty and Cunningham, 1975) and transgenic mouse studies of first-generation CARs (Brocker, 2000). These findings prompted us to combine chimeric costimulatory receptors that we had previously studied in human primary T cells (Krause et al., 1998) with T-cell-activating CARs, thus creating dual-signaling receptors (Figure 3C) that we found were able to instruct human peripheral blood T cells to expand and retain function upon repeated exposure to antigen (Maher et al., 2002). These and other contemporary studies (Finney et al., 1998; Hombach et al., 2001; Imai et al., 2004) paved the way for engineering persisting functional T cells, which we later named ‘‘living drugs.’’ Second-generation CARs are a new class of drugs with exciting potential for cancer immunotherapy (Sadelain et al., 2013; Jensen and Riddell, 2015). Recent clinical results obtained with CD19 CARs are reviewed in Ramos et al. (2014) and Kochenderfer and Rosenberg (2013). CAR therapy promises to produce more durable cancer regression in other hematological malignancies and solid tumors, including those that do not present altered self-antigens. CARs may be combined with costimulatory ligands (Stephan et al., 2007), chimeric costimulatory receptors (Krause et al., 1998; Duong et al., 2011; Wilkie et al., 2012; Prosser et al., 2012; Kloss et al., 2013), or cytokines such as IL-15 (Hoyos et al., 2010; Markley and Sadelain, 2010) or IL-12 (Chinnasamy et al., 2012; Chmielewski and Abken, 2012; Pegram et al., Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc. 7

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Perspective 2012) to further enhance T cell potency, specificity, or safety. Unlike CARs, CCRs do not trigger T cell activation, but rather offset apoptosis and extend T cell persistence in antigen-specific fashion (Krause et al., 1998). Antigen-specific inhibitory receptors, such as iCARs modeled on CTLA-4 and PD-1 (Fedorov et al., 2013), further extend this growing panoply of recombinant receptors. In a futuristic embodiment, bringing together thymopoiesis and cell engineering (Zhao et al., 2007; Themeli et al., 2013), the ability to recapitulate thymic maturation in vitro and generate T cells from pluripotent stem cells (Nishimura et al., 2013; Vizcardo et al., 2013; Themeli et al., 2013) may open a path to design and manufacture therapeutic T cells with broad histocompatibility and optimized functional features (Themeli et al., 2015). Perspective The spectacular successes that have been achieved in the immune management of various clinical conditions and especially cancer were borne out of basic research that was creatively exploited by translational researchers. No one could have predicted that investigating how or why virus-induced lymphocytic leukemia needs to develop in the neonatal mouse thymus would reveal the latter’s immunological function. The extensive worldwide research that followed was crucial to our understanding of what cells and molecules regulate T cell activation and how this may be used to our benefit in the clinic. The advent of monoclonal antibody and gene transfer technologies later enabled creative exploitation of fundamental knowledge on T and B cell antigen recognition, T cell activation, and T cell costimulation, leading to the invention of checkpoint blockade and CAR T cell therapy. While some key molecular pathways and cellular interactions have been decoded, much more is yet to be discovered. More immunology research is warranted. In the meantime, immunology has spawned immunotherapy, which is about to claim a seat in the therapeutic pantheon of oncology, next to surgery, radiation therapy, and chemotherapy. ACKNOWLEDGMENTS We thank Drs. Tony Basten, Marc Feldmann, Dale Godfrey, Robyn Slattery, and Andreas Strasser for reading the manuscript and for helpful suggestions. REFERENCES Abu-Eid, R., Samara, R.N., Ozbun, L., Abdalla, M.Y., Berzofsky, J.A., Friedman, K.M., Mkrtichyan, M., and Khleif, S.N. (2014). Selective inhibition of regulatory T cells by targeting the PI3K-Akt pathway. Cancer Immunol Res 2, 1080–1089. Alexandrov, L.B., Nik-Zainal, S., Wedge, D.C., Aparicio, S.A., Behjati, S., Biankin, A.V., Bignell, G.R., Bolli, N., Borg, A., Børresen-Dale, A.L., et al.; Australian Pancreatic Cancer Genome Initiative; ICGC Breast Cancer Consortium; ICGC MMML-Seq Consortium; ICGC PedBrain (2013). Signatures of mutational processes in human cancer. Nature 500, 415–421. Asseman, C., Mauze, S., Leach, M.W., Coffman, R.L., and Powrie, F. (1999). An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190, 995–1004.

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