Targeting Neoantigens in Glioblastoma

Abstract

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 extracapillary layer of cells including pericytes and astrocytic end-foot processes, which form the glia limitans (Figure 2). It is this dual layered barrier that restricts transfer of solute and cells into the brain parenchyma. However, in GBM, this barrier is compromised (Figure 2). For example, Nduom and colleagues33 observed by immunohistochemistry that in patients with GBM, regions of magnetic resonance imaging (MRI) enhancement corresponded with breakdown of the normal astrocyte-endothelial cell relationship demonstrated by gaps between GFAP (glial fibrillary acidic proteins; expressed by astrocytic cells) and the aquaporin molecule, AQP4, which is expressed on the luminal side of glial processes. These proteins should normally demarcate a tight boundary on the basolateral side of astrocytes that surround the endothelial cells to secure the BBB as demonstrated in nonenhancing lesions.33 A similar phenomenon has been observed in brain metastases and pediatric high-grade gliomas, but not in low-grade, non-MRI-enhancing tumors.33-35FIGURE 2: Proposed model of leukocyte recruitment due to altered BBB integrity in GBM. A, Under normal conditions, the dual layers of the BBB is maintained through tight junctions between capillary endothelial cells and the glia limitans, which is comprised of astrocytic end-foot processes. In the postcapillary venules, these 2 layers separate creating a perivascular (Virchow-Robin) space, which contains resident macrophages. B, In the context of GBM or inflammation, the BBB is disrupted. The glia limitans loses polarity due to altered expression of AQP4 in the astrocytic end-foot processes leading to expansion of the perivascular space and communication with the underlying parenchyma (1). The capillary tight junctions are disrupted due to reduced expression of claudin-3, which permits exchange of solutes, antigens, and chemokines/cytokines (2). It also allows circulating leukocytes, such as neutrophils, monocytes, and T cells, to gain access to the perivascular space where they interact with APC that present tumor antigen from the parenchyma (3). AQP4, aquaporin 4 molecule; BBB, blood–brain barrier; GBM, glioblastoma.In addition to disruption of the BBB due to altered polarity of the astrocytic end-foot processes, the endothelial layer is also perturbed. Particularly, the interendothelial cell tight junctions, which are essential to maintaining the integrity of the BBB, also becomes dysregulated in GBM. Wolburg et al35,36 observed a loss of the tight junction molecule, claudin-3, in GBM, which greatly contributed to the increased permeability noted in intratumoral capillary vessels. Together, these findings demonstrate a dramatic loss of integrity of the BBB adjacent to GBM that affects both the endothelial layer and glia limitans (Figure 2). What is interesting to note is that the perivascular space, which forms between the endothelial cell layer and the glia limitans in the postcapillary venules, becomes expanded at the site of BBB disruption in GBM.35,36 The perivascular space is also the site of resident macrophages. Therefore, one potential mechanism by which a local immune reaction is incited against GBM is that the breakdown of the BBB facilitates detection by and activation of resident macrophages to GBM-associated antigens leading to recruitment of circulating immune cells that are then able to recognize cognate antigens on local antigen-presenting cells(APC) (Figure 2). Consistent with this model, Proescholdt et al37 noted that it is not until the BBB is disrupted that an immune infiltrate is detected in a rat brain tumor model. CANCER IMMUNOGENOMICS AND THE IDENTIFICATION OF NEOANTIGENS IN GBM Cancer immunogenomics represents a complementary approach to the application of genomics in developing novel treatment strategies for malignancies. Using this approach, putative tumor-specific neoantigens derived from expressed, nonsynonymous missense or frameshift mutations in the exome are prioritized based on predicted processing and binding affinity to a patient's individual HLA (human leukocyte antigen) molecules.38 Thus, rather than stratifying mutational targets based on the "drivers" and "passengers" classification, the predicted immunodominance of a mutational alteration is given precedence, creating a "mutation-to-antigenic target" paradigm. This approach is increasingly being applied to neoantigen identification both preclinically and clinically. The actual process of neoantigen discovery using this approach will be discussed here. Definition of Neoantigen We now know that endogenous T cells recognize tumor antigens presented by major histocompatibility complexes (MHC) on the surface of malignant cells. These recognition events are mediated by specific interactions between MHC-bound tumor antigens and T cell receptors.39 To date, 3 classes of MHC-binding tumor antigens have been documented40: (1) shared tumor antigens which are nonmutant, normally expressed proteins that are aberrantly overexpressed in tumor cells, (2) cancer-testis antigens that are normally only found in healthy adult germ cell tissues but exhibit re-expression in some cancers, and (3) tumor-specific mutant antigens, referred to as neoantigens, which represent novel peptide sequences encoded by somatic mutations in the cancer genome. To date, cancer vaccine clinical trials that have used peptide-based vaccines comprising of shared-tumor antigens or cancer-testis antigens have not yielded promising results despite concomitant induction of a high frequency of antigen-specific T cells.41 One theory underlying the lack of success may be attributable to issues of central tolerance whereby high-affinity endogenous T cells specific to these conserved tumor antigens are eliminated due to expression in normal tissue during development. Additional challenges common to both types of antigens include limited expression in tumor cells compared to levels in nonmalignant cells; lack of known binding within less common HLA alleles precluding their broader use in many patients; as well as increased risks of "off-target" immune recognition of nonmalignant cells. Compared to nonmutant tumor-associated antigens, neoantigens circumvent issues of immune tolerance as they consist of peptides derived from somatic, nonsynonymous mutations only present in the tumor genome, and therefore would appear as "foreign" to the host immune system. Likewise, immunodominant neoantigens are tailored to a patient's specific HLA alleles, permitting the broader application of this approach to a larger, more diverse patient population.38 Cancer Immunogenomics: Pipeline for Neoantigen Discovery Cancer immunogenomics refers to a concept in which genomic alterations inherent to cancer cells are leveraged as targets for immune-based therapies.42-45 One example of this strategy is the identification and targeting of neoantigens. Until recently, the identification of patient tumor-specific neoantigens required highly labor-intensive laboratory techniques that precluded its use in the clinical setting. However, the development of next-generation sequencing technologies and advances in the downstream computational analyses have revitalized these efforts by facilitating rapid characterization of the tumor mutational landscape. By removing these technological barriers, genomic breakthroughs have paved the way for high-throughput and cost-effective personalized neoantigen identification. The initial step in identifying neoantigens begins with DNA whole exome and RNA sequencing of matched patient normal and tumor tissue (Figure 3). Using one of many currently available variant calling and annotation software programs, the raw exome sequence data are mined for nonsynonymous missense tumor variants and integrated with transcriptome analysis to select for expressed mutations.46 Peptide sequences containing the encoded amino acid mutations are then generated according to predesignated residue length settings to accommodate the different binding grooves of MHC class I or class II molecules. Due to the vast number of candidate neoantigen peptides that can be generated for a given tumor, in silico algorithms are used to aid in the selection of immunogenic neoantigens by predicting the binding affinity of each candidate peptide for patient-specific HLA alleles. Ultimately, candidate peptides with the highest predicted binding affinity are synthesized and used in either personalized vaccines or in a variety of immunological assays to validate the presence/generation of neoantigen-specific T cell responses in an individual (Figure 3).47,48FIGURE 3: Schematic representation of cancer immunogenomics workflow for neoantigen discovery. Normal reference tissue (ie, PBMC) and tumor tissue is obtained and undergoes DNA whole exome and RNA sequencing to identify somatic, nonsynonymous mutations. Tumor-specific mutations are then filtered using computational software to prioritize neoantigens based on expression, predicted patient-specific HLA binding affinity, and likelihood of endogenous proteosomal processing. Peptides corresponding to candidate high-quality neoantigens are then manufactured and administered back to the patient as a personalized vaccine. PBMC, peripheral blood of The computational analysis in the context of neoantigen discovery can be thought of as occurring in 2 with the initial processing of raw genomic data and into the use of in silico immunogenomics to the cell A number of different software are available for the initial processing of cancer sequence These include to identify or as well as annotation algorithms that alterations in and based on genomic and the functional annotation of a variant on the or For example, the most used annotation include and which of used to the functional of a given in an analysis by and the found that using different results in variant annotation Thus, as variant annotation is not yet this step must be in the broader of neoantigen identification. for MHC I In the of neoantigen mutant peptide sequences generated from genomic analyses are filtered in order to select only for that are likely to elicit T cell responses. To aid in the selection of immunogenic neoantigens from that often of candidate in silico algorithms are used to peptide binding for patient-specific MHC less in predicting peptide binding in is more given the multiple HLA alleles present in a given on the HLA is the most regions in the human genome, with current HLA comprising distinct alleles. 3 HLA class I and and 3 HLA class II from each each can to HLA each capable of a distinct of neoantigens. As such, this must be by algorithms in order to prioritize that could ultimately be on a specific patient's MHC molecules. the vast majority of computational for predicting neoantigen binding affinity use a of and The such as and binding using position-specific scoring and therefore the of these on the of data for specific MHC such as the Immune have to by multiple different over the use of is contains over and for this reason is used for neoantigen are limited by their on available data and therefore when predicting binding for less common HLA alleles, which is known Therefore, new computational such as have been that peptide binding affinity for any MHC without the need for These are on that are on available binding data and then to novel binding While still limited by the size and of in have been by to include from there is no on for specific neoantigen results from a number of recent studies such as the highest For example, and a analysis to the of algorithms to immunogenic neoantigens from on a of immunogenic neoantigens, of the naturally occurring to be immunogenic based on the binding affinity of However, the most in the antigen binding for of the immunogenic neoantigens, as a number of affinity, immunogenic peptides have been MHC Compared to in silico of MHC class there has been less in developing computational for predicting interactions with MHC class II molecules. MHC class algorithms and the in neoantigen discovery has been on class I and the class II algorithms lack MHC class II peptide will also likely a number of to address the between the 2 MHC MHC class II are only expressed on APC such as dendritic cells, and B cells, and play an role in T cells. also to of the peptides that each Unlike MHC class I the MHC class II is at both such that peptides of to amino can This in both the length of peptides and in the of the as the peptide is to the binding The a as class II binding affinity is not only by binding sequence but also by Thus, due to the of class II interactions and the lack of available class II peptide algorithms are less than for class I the new efforts are being in class II in of recent studies demonstrating the contribution of CD4+ T cells in antitumor A by and that T cell responses to neoantigen in mice found that of the neoantigen-specific T cells were of this phenomenon in human malignancies resulted in the that with CD4+ T cells specific for an neoantigen tumor in a patient with in patients a of neoantigen-specific intratumoral CD4+ T Together, these clinical data support the need to to class neoantigen as class II antigens the of potential targets that can be into personalized which may have for patients with GBM that a mutational to in silico Neoantigen While current computational algorithms MHC class events with high this other in the antigen pathway including peptide processing. These including and of peptides via proteins into the also which putative neoantigens are ultimately presented by MHC new computational have been that the of peptide by the and interactions with in order to that may be in identifying naturally and a similar to class I binding affinity and to identify neoantigens. a of immunogenic neoantigens, of neoantigens, out of neoantigens. Furthermore, the the use of a and found that of the neoantigens mutations within the of the These data that current in neoantigen will likely be by the development of computational that a understanding of the antigen While of current in silico neoantigen have greatly improved the of peptide-based have to in order to circumvent inherent challenges with HLA and endogenous peptide processing. and a novel tandem minigene approach to identify neoantigens by T cells in 2 patients had following TIL