Targeting Neoantigens in Glioblastoma

BBB: blood–brain barrier CNS: central nervous system CSF: cerebrospinal fluid EGFRvIII: epidermal growth factor receptor variant III GBM: glioblastoma MHC: major histocompatibility complexes MRI: magnetic resonance imaging PSSM: position-specific scoring matrix TCGA: The Cancer Genome Atlas TIL: tumor-infiltrating lymphocytes TMG: tandem minigene Glioblastoma (GBM) remains a disease with a poor prognosis. Unfortunately, over the past decade, no new treatment options have improved survival in patients beyond the current standard-of-care radiation plus temozolomide following maximal surgical resection. The initial enthusiasm that extensive genomic profiling of driver mutations, of which GBM was one of the first to be characterized by The Cancer Genome Atlas (TCGA),1,2 would lead to effective molecularly targeted therapy for central nervous system (CNS) malignancies has yet to come to fruition. The reason for the failure of this “mutation-to-drug” paradigm is likely multifactorial, including the subclonal heterogeneity of GBM3 and the necessity of systemically delivered drugs to penetrate the blood–brain barrier(BBB) at a sufficient concentration to be efficacious. As such, any new treatment approach will need to address these complexities of GBM if it is to be successful. It is to this end that immunotherapy offers renewed promise. The systemic immune system has the ability to attack multiple targets simultaneously, and has the capacity to penetrate the BBB. As our understanding of CNS immunosurveillance and tumor immunity continues to deepen, novel strategies to prime and augment a potent antitumor immune response will emerge. Recent interest has been focused on the identification of tumor-specific mutations, termed neoantigens, which can serve as immunodominant targets for antitumor immune effector cells to maximize “on-tumor” effect and minimize “off-tumor” toxicities. In this review, we will discuss: (1) the current perspective on CNS immunosurveillance, (2) the process of neoantigen identification focusing on the cancer immunogenomics approach, and (3) how this strategy can be used to target GBM specifically. EVIDENCE OF ACTIVE IMMUNOSURVEILLANCE IN GBM: CNS IMMUNOBIOLOGY The potential of immunotherapy in CNS malignancies has long been thought to be futile given the immunoprivileged and immunosuppressive nature of the intracranial environment. However, recent data have demonstrated that the CNS is not wholly a sanctuary site due to immune isolationism. On the contrary, the immune system actively surveys the CNS, and is capable of mounting an effective immunological response when necessary supporting the renewed enthusiasm for immunotherapy in combating CNS disease. Immunoprivilege in the CNS The topic of CNS immunosurveillance has been extensively reviewed recently,4-8 and is not within the scope of this article. However, given that the historic viewpoint of an “immunoprivileged CNS” has often been interpreted as an “immunocompromised CNS,” several key concepts must be discussed in order to understand the rationale for pursuing immune therapy in GBM. As summarized eloquently by Engelhardt and colleagues,4 the immunoprivileged phenotype of the CNS was based on the experimental observation that tissues grafted into the brain parenchyma are not rejected due to the lack of an induced cell-mediated immunity.9 Importantly, the simultaneous implantation of skin homografts subcutaneously led to equivalent rejection of both the skin and brain grafts9 implying that the effector arm of the systemic immune system is able to sufficiently locate, penetrate, and remove CNS-based antigens. Similar results were demonstrated following intraparenchymal injection of bacillus Calmette-Guerin that resulted in a demyelinating delayed type hypersensitivity reaction following subsequent systemic immunization despite a minimal local reaction initially.10 These experiments support the notion that a deficient afferent limb of the immune response may be largely responsible for the immunoprivileged phenotype of the brain parenchyma, and that the effector arm is functionally intact. Importantly, it should be noted that this observation is perhaps most relevant under steady state circumstances. For instance, the intraparenchymal injection of immunostimulatory agents such as lipopolysaccharide,11 TNFα, or IL-1αβ12 leads to robust, albeit delayed, influx of innate immune cells such as neutrophils, monocytes, and macrophages as well as activation of resident microglial cells demonstrating that local inflammation does indeed drive recruitment and infiltration of systemic immune cells. Therefore, under inflammatory conditions, the immunoprivileged nature of the brain parenchyma is subverted.5 Furthermore, it should also be pointed out that these observations are limited to the brain parenchyma as implantation of virus or tissue grafts into the cerebrospinal fluid (CSF) or choroidal plexus results in robust immune responses equivalent to systemic sites.13-15 Mechanisms of CNS Antigen Drainage to Lymph Nodes The apparent deficiency of the afferent limb within the brain parenchyma was initially attributed to the presence of the BBB and lack of classic lymphatics. While the CNS is certainly immunologically distinct from other organs anatomically, the immune system is still able to actively survey CNS antigens through various mechanisms. Specifically, there are 3 primary routes by which intracranial antigens, and presumably tumor-derived antigens, are able to drain from the CNS into locoregional and systemic lymphoid tissue (Figure 1). The first is via ventricular and subarachnoid CSF that is able to cross the cribiform plate and enter the lymphatics of the nasal mucosal ultimately draining into the deep cervical lymph nodes.7 Secondly, CSF is able to enter recently described meningeal lymphatics located in the dura that also drain to the deep cervical lymph node chain.16,17 These routes of CNS drainage are amenable both to soluble antigens as well as immune cells such as T cells, monocytes, and dendritic cells. The third route CNS-derived antigens use to reach regional lymph nodes results from parenchymal interstitial fluid trafficking through the basement membrane of the wall of capillaries and arteries of the brain.18 Unlike the CSF, arterial-based drainage is limited to acellular antigen transportation due to size exclusion. Alternatively, intraparenchymal interstitial fluid is also exchanged with CSF in a process termed glymphatics.19 It is interesting to note that drainage of parenchymal antigens is abrogated in mice lacking meningeal lymphatics16 despite only ∼15% of interstitial fluid from the parenchyma draining through the CSF.20 Therefore, it seems reasonable to assume that GBM-derived antigens are able to reach draining lymph nodes via both routes, though the relative contribution in human disease remains unclear and may be largely dependent on geography of the tumor.FIGURE 1: Routes of CNS-based tumor antigen drainage to regional lymph nodes. Tumor-derived antigens can reach draining cervical lymph nodes in several ways. Antigen that gains access to the CSF either by direct extension of the tumor, breakdown of the BBB, cellular trafficking by APC, or through glymphatic exchange can enter the lymphatic system by traversing the cribiform plate into the nasal mucosa (1) or through meningeal lymphatics of the dura (3). Alternatively, acellular antigen can enter the wall of intraparenchymal capillaries and arteries to migrate retrograde toward local lymph nodes (2). BBB, blood–brain barrier; CSF, cerebrospinal fluid; GBM, glioblastoma; ISF, interstitial fluid. Adapted from Engelhardtet al.4Effector Immune Responses to GBM Regardless of the pathway used by GBM-derived antigens to translocate to local draining lymph nodes, it is clear that such antigens are able to elicit effector immune responses. For example, spontaneously arising autoantibodies to GBM-specific proteins: GLEA1, GLEA2, and PHF3 have been demonstrated in 24%, 48%, and 57% of adult GBM patients, respectively,21 providing support for the generation of a naturally occurring antiglioma humoral response. Likewise, the cellular arm of the immune system also appears to be primed against GBM tumor cells. Barcia et al22 observed activated cytotoxic CD8+ T cells in close proximity to GBM tumor cells in Situ, characterized by CD3/T cell receptor (TCR) clusters, cytoskeletal rearrangement, and granzyme B polarization toward the tumor cells supporting recognition of cognate antigen:MHC complexes on GBM cells by antigen-specific T cells. Additionally, Berghoff and colleagues23 reported that the majority of newly diagnosed patients (72.6%) and recurrent patients (83.3%) had tumor-infiltrating lymphocytes (TILs) present in tumor specimens, indirectly pointing toward an interaction between tumor and the host immune system. While TILs are largely confined to the perivascular space of postcapillary venules and peripheral zones of tumor invasion,23 numerous studies have demonstrated a positive correlation between the presence of TIL and clinical outcome for patients with GBM.24 For example, Brooks et al25 examined clinical records and biopsy specimens of 149 patients from 1962 to 1976 and noted that perivascular lymphocyte infiltration correlated with a 2 to 4 mo increase in survival over patients without such infiltrate. Obviously, TIL represent a heterogeneous group of immune cells, comprising both effector and suppressive subsets. Thus, as one might expect, the effect on survival largely seems to be dependent upon the ratio of effector T cells (ie, CD4+ or CD8+ subsets) to suppressor T cells (Tregs).26-30 Together, these data support the notion that both the humoral and cellular arms of the immune system are able to be primed against GBM antigens. However, it remains unclear by which mechanism these adaptive immune responses are generated; which antigens they recognize; the functional capacity of such naturally occurring responses; and the role these spontaneous immune responses play in driving immune escape. THE BBB IN GBM The exact mechanism leading to recruitment of infiltrating lymphocytes into the GBM microenvironment is not understood. One potential explanation is that the BBB is compromised in the setting of GBM, and this “leaky” BBB could serve as a conduit for interactions between lymphocytes and the tumor.31,32 At steady state, the BBB is composed of tight junctions between specialized capillary endothelial cells supported by an of cells including and which the (Figure It is this barrier that of and cells into the brain However, in GBM, this barrier is compromised (Figure For example, and observed by that in patients with GBM, of magnetic resonance imaging with breakdown of the cell demonstrated by between by and the which is on the of These should a tight on the of that the endothelial cells to the BBB as demonstrated in has been observed in brain and not in of recruitment due to BBB in GBM. conditions, the of the BBB is through tight junctions between capillary endothelial cells and the which is of In the postcapillary these 2 a perivascular which resident In the of GBM or the BBB is The due to of in the leading to of the perivascular space and with the parenchyma The capillary tight junctions are due to of which exchange of antigens, and (2). It also such as neutrophils, monocytes, and T cells, to access to the perivascular space they with that present tumor antigen from the parenchyma (3). 4 BBB, blood–brain barrier; GBM, to of the BBB due to of the the endothelial is also the cell tight which are to the of the BBB, also in GBM. et observed a of the tight in GBM, which to the noted in capillary Together, these a of of the BBB to GBM that both the endothelial and (Figure is interesting to note is that the perivascular which between the endothelial cell and the in the postcapillary at the site of BBB in The perivascular space is also the site of resident Therefore, one potential mechanism by which a local immune reaction is against GBM is that the breakdown of the BBB by and activation of resident macrophages to antigens leading to recruitment of immune cells that are able to cognate antigens on local (Figure with this et noted that it is not the BBB is that an immune is in a brain tumor THE OF IN GBM Cancer immunogenomics a approach to the of in novel treatment strategies for this approach, tumor-specific from or in the are based on and to a Thus, targets based on the and the of a is given a approach is to neoantigen identification both and The process of neoantigen this approach will be discussed of that T cells tumor antigens by major histocompatibility complexes on the of cells. These recognition are by interactions between tumor antigens and T cell 3 of tumor antigens have been (1) tumor antigens which are that are in tumor cells, (2) antigens that are only in adult cell tissues in and (3) tumor-specific antigens, to as neoantigens, which represent novel by in the cancer cancer clinical that have used comprising of antigens or antigens have not results despite of a of antigen-specific T One the lack of may be to of central T cells to these tumor antigens are due to in tissue to both of antigens limited in tumor cells to in lack of within use in as well as of immune recognition of cells. to antigens, of immune as they of from only present in the tumor and would as to the host immune system. Likewise, immunodominant are to a the of this approach to a Cancer for Cancer immunogenomics to a in which genomic to cancer cells are as targets for One of this strategy is the identification and of the identification of tumor-specific that use in the clinical However, the of and in the have these by of the tumor these genomic have the for and neoantigen The initial in with and of and tumor tissue (Figure one of variant and the data are for tumor and with to for the are to to the of or to the of neoantigen that can be for a given tumor, in are used to in the of by the of for with the are and used in either or in a of immunological to the of T cell responses in an (Figure of cancer immunogenomics for neoantigen tissue (ie, and tumor tissue is and and to are to based on and of to are and to the as a peripheral of The in the of neoantigen can be thought of as occurring in 2 with the initial of genomic data and into the use of in immunogenomics to the cell of are for the initial of cancer These to or as well as that in and based on genomic and the functional of a variant on the or For example, the most used and which of used to the functional of a given in an by and the that results in variant Thus, as variant is not yet this must be in the of neoantigen for In the of neoantigen from genomic are in order to only for that are likely to elicit T cell responses. in the of from that often of in are used to for in in is given the multiple present in a given on the is the most in the human with current comprising distinct 3 and and 3 from can to capable of a distinct of As such, this must be by in order to that could ultimately be on a the majority of for neoantigen use a of and The such as and position-specific scoring and the of these on the of data for such as the Immune have to by multiple over the use of is over and for this reason is used for neoantigen are limited by on data and when for which is Therefore, new such as have been that for any without the need for These are on that are on data and to novel While still limited by the size and of in have been by to from there is no on for neoantigen results from a of recent studies such as the For example, and a to the of to from on a of neoantigens, of the naturally occurring to be based on the of However, the most in the antigen for of the neoantigens, as a of have been to in of there has been in for interactions with and the in neoantigen has been on and the lack will also likely a of to address the between the 2 are only on such as dendritic cells, and B cells, and play an role in T cells. also to of the that Unlike the is at both such that of to can in both the of and in the of the as the is to the The a as is not only by also by Thus, due to the of interactions and the lack of are for the new are in in of recent studies demonstrating the contribution of CD4+ T cells in antitumor by and that T cell responses to neoantigen in mice that of the T cells were of this in human malignancies resulted in the that with CD4+ T cells for an neoantigen tumor in a with in patients a of CD4+ T Together, these clinical data support the need to to neoantigen as antigens the of potential targets that can be into which may have for patients with GBM that a to in While current with this other in the antigen pathway including These including and of via into the also which are ultimately by new have been that the of by the and interactions with in order to that may be in naturally and a to and to a of neoantigens, of neoantigens, out of Furthermore, the the use of a and that of the within the of the These data that current in neoantigen will likely be by the of that a understanding of the antigen While of current in neoantigen have improved the of have to in order to with and and a novel tandem minigene approach to by T cells in 2 patients had following TIL In to tumor were into In to T cell response to were and subsequent of T cell recognition and as TIL These results the of a approach for neoantigen studies will be to it to Recent in the of have in neoantigen as a to in interactions on the tumor cell In are by of to be ultimately are not in to of of studies have recently to use an approach that these 2 for neoantigen and demonstrated that with 2 by both in and resulted in T cell responses in and neoantigen by with by in patients with of the by were the as by though 2 of the were to elicit T cell responses. These studies that may of in for the data represent tumor heterogeneity studies in tumor cell and also of in neoantigen that is from Thus, of data into neoantigen identification to of and the of While the of by in to the by in may be attributed to of also with this observation that for of and may in of the neoantigen clinical use of this is limited by the tissue size for as well as the to such However, as in and to robust results from experiments will likely the generation of neoantigen IN GBM The of neoantigen is to target various The of and has the of on a from both a and Importantly, the of such an approach have been demonstrated and have 2 patients, with and the other with an cell approach with TIL to The targeted a and a In both the CD8+ or CD4+ T cells resulted in of the disease. a of are to to patients with malignancies from various including GBM, multiple including and dendritic several have been targeted in GBM and to III growth factor receptor variant III is a of that is present in to of from an of within the of The the a neoantigen and a this novel was the of resulted in in survival for patients with GBM in studies to historic the recent III to a survival despite a effect on survival as with the in The was due to a survival in the key from the is to be in only a of tumor and of has been in that a subclonal neoantigen may be due to on Alternatively, it may be necessary to target multiple subclonal if a neoantigen is not is that patients were given of As such, studies largely demonstrated a humoral response a cell-mediated Thus, it is that a cell-mediated immune response would be in which which are capable of may lead to improved the results from and results into in GBM in the of which over a represent an In are thought to be an are present in the and by tumor cells. this is in the majority of and a of Furthermore, et demonstrated both cellular and humoral immune responses to an long in patients with that it is a potential in that are to with to the providing that are an immunodominant target of CD4+ T cells. these data not only support for pursuing the neoantigen as a target also the potential of neoantigen in to the of as and as for will on as well as For example, in the of or GBM there are no likely targets will be from subclonal In the and of with was demonstrated by et in which the of a mice against a As for this in GBM, we recently reported the identification of in the cancer immunogenomics approach described in approach initially a of in both the and spontaneously a strategy that and we 2 neoantigens, and as and capable of spontaneous T cell responses both in and draining lymph nodes. Importantly, of the between these 2 these were the only 2 that were as immunodominant this is to other and clinical studies multiple tumor As such, this the need to upon the current in used to in an to increase the of neoantigen Furthermore, it rationale for a approach, as it will increase the of with a the cancer immunogenomics to for patients with GBM are One of the major in these studies from the and of this approach will be to the of given the in GBM. et a neoantigen to a of GBM in the and that on these from which potential were The from this to note is that the neoantigen were limited to Thus, the of a to would the of potential which may be in these the of CD4+ T cells observed by and also for focusing on in these and other studies will the of neoantigen a must be on the studies to how effective these are at immune responses most disease. from as targets for clinical for the neoantigen of is that a neoantigen can also serve as a for to other of such as was initially reported by and in cancer patients, they demonstrated a response to therapy in patients were deficient in the pathway to with a correlation between and by extension neoantigen with response to has been demonstrated in cell and The use of neoantigen as a to patients that may from therapy also appears to to patients with GBM. In 2 recent both an adult with a GBM to a as well as 2 patients with GBM due to were to have responses to therapy on of While the of patients with GBM is at of there is that a of recurrent GBM, to a phenotype at of due to an deficiency in the seems to be in patients with for and with Together, these data would a treatment strategy in GBM patients with neoantigen would be to therapy and with neoantigen could a that clinical are necessary to the for immunotherapy and cancer immunogenomics into the treatment of GBM. a approach to GBM, as well as other as it is distinct from of the nature of the immune system to target tumor-specific neoantigens, immunotherapy offers an strategy to maximize “on-tumor” the recent in genomic and the of has a the clinical such strategies has both the enthusiasm and in these treatment for a of cancer However, a of studies will be at (1) to the through novel and to increase the of neoantigen (2) understanding of or (3) immunization in order to the most effective immune response to and how to or immunotherapy with current cytotoxic and to the cancer treatment options is a of was by of Cancer the and the at of The have no or interest in any of the or described in this article.