Thesis
Reference
Improved glioblastoma mouse models to design better immunotherapeutic combinations
GENOUD, Vassilis
Abstract
Le glioblastome est le cancer primaire du cerveau le plus fréquent, et malgré les traitements actuels, le pronostique reste très limité. Toutefois pour d'autres cancers nous notons une nette avancée de l'efficacité des traitements avec l'utilisation des inhibiteurs de points de contrôles immunitaires (IPC). En revanche les premiers résultats d'études clinique semblent indiquer une résistance du glioblastome à cette thérapie. Dans ce travail, nous étudions plusieurs modèles murins du glioblastome afin qu'ils soient les plus représentatifs des caractéristiques humaines impliquées dans la réponse aux IPC. En particulier le nombre total de mutation et l'infiltration tumorale de cellules T, qui sont directement liées à la réponse aux IPC, et nous pouvons modéliser la résistance aux IPC dans un modèle murin. Nous identifions ensuite une combinaison de chimiothérapie et de stimulateur du système immunitaire inné, à savoir du récepteur CD40, qui s'avère plus efficace que les IPC pour ce type de tumeur.
GENOUD, Vassilis. Improved glioblastoma mouse models to design better
immunotherapeutic combinations . Thèse de doctorat : Univ. Genève, 2019, no. Sc. Méd.
37
DOI : 10.13097/archive-ouverte/unige:129407 URN : urn:nbn:ch:unige-1294072
Available at:
http://archive-ouverte.unige.ch/unige:129407
Improved glioblastoma mouse models to design better immunotherapeutic
combinations
THESE D E D OCTOR AT MD -PhD
Présenté à la faculté de Médecine de l’Université de Genève Par
VASSIL IS G ENO UD
De Bagnes (VS)
Thèse n°37 JURY
Dr. Paul R Walker, co-direction de thèse Prof. Pierre-Yves Dietrich, co-direction de thèse Prof. Doron Merkler, membre du comité de thèse Prof. Jean Villard, représentant du comité scientifique
Prof. Olivier Michielin, expert externe
Genève 2019
Table of contents
1. Thesis abstract ... 4
2. General introduction ... 7
a. Brain tumors ... 7
b. Animal models ... 9
c. The immune system ... 10
i. Innate immunity ... 10
ii. Adaptive immunity ... 11
iii. Brain particularities ... 17
d. Immuno-Tumoral interactions ... 18
i. Cancer-immunity cycle ... 18
ii. Immunoediting ... 19
iii. Glioma escape ... 19
iv. Immune context... 21
e. Immunotherapy for cancer ... 21
i. Vaccination ... 22
ii. Adoptive cellular therapies ... 23
iii. Therapies using antibodies ... 24
iv. Immunomodulatory antibodies ... 24
v. Other immunotherapeutic approaches ... 26
vi. Immunotherapeutic combinations ... 27
3. Objectives ... 31
4. Projects and publications ... 32
a. Chapter 1 – Glioma mouse model characteristics and their predictive value regarding ICB sensitivity ... 32
b. Chapter 2 – Combination of immunotherapy and chemotherapy for an ICB resistant GBM mouse model ... 43
5. Additional data ... 84
b. Subcutaneous SB28 implantation ... 85
6. Discussion and perspectives ... 88
a. Biomarkers ... 88
b. Immunogenicity ... 91
c. Treatment strategies ... 93
7. Conclusion ... 95
8. Acknowledgments ... 98
9. List of Abbreviations ... 99
10. References ... 101
1. Thesis abstract
BACKGROUND. Glioblastoma (GBM) is the most frequent primary brain tumor. Standard of care treatment consists of surgery followed by radio-chemotherapy, but offers a poor prognosis with only 10% of survival at 5 years and no improvements were brought to patients in the last decade. On the other hand, for several other cancer indications, new immunotherapeutic approaches have proven their efficacy, notably immune checkpoint blockade (ICB) comprised of antagonistic antibodies (Ab) to Programmed cell death 1 (PD-1) and its ligand (PD-L1) and/or Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) that enhance anti-tumor immunity at different steps of the anti-tumor immune response.
Nevertheless, only certain patients benefit from ICB, particularly those with tumors that have a high mutational load and which are well-infiltrated with immune cells. When envisioning ICB for GBM, the particularities of the brain tumor site must be considered.
Current interpretations of the concept of immune privilege of the brain are consistent with some protective intracranial immune reactions, which may be subject to additional regulation to avoid excessive inflammation and neurological damage. Nevertheless, clinical data confirms positive responses to ICB for certain brain metastases, in rare cases of hypermutated GBM and also recently in neoadjuvant therapy for recurrent GBM.
Conversely, other preliminary results from ICB monotherapy of patients with primary GBM indicate resistance. Considering current hypotheses concerning mutational load, and decades of research describing immunosuppressive cells and factors in GBM, it is clear that multiple factors could account for this heterogeneity in responsiveness. In view of the plethora of immunotherapeutic tools being developed for cancer, to rationally select appropriate agents and combinations for GBM we require more insight into GBM responsiveness and non-responsiveness to immunotherapy and other compatible treatment modalities.
AIMS.
1. To assess whether proposed prognostic indicators of responsiveness to PD-1/PD-L1 and CTLA-4 blockade can be extended to two syngeneic mouse GBM models.
2. To test whether alternative immunomodulators to ICB, and new-generation chemotherapies can be considered for categories of GBM that are ICB-resistant.
METHODS. We characterized two mouse GBM models that were developped by different technologies and that were predicted to differ in mutational load: the carcinogen induced GL261 model and the SB28 model genetically engineered to target defined oncogenes and tumor suppressors. We studied phenotype by flow cytometry and mutational load by Next-Generation Sequencing (NGS). The two models were ultimately compared head to head for their response to a clinically relevant combination of anti-PD- 1 and anti-CTLA-4 antagonistic Ab. We also tested other treatment modalities, namely chemotherapy, radiotherapy, and an alternative immunomodulator comprised of agonistic anti-CD40. Survival increase and modulation of immune infiltration was assessed by flow cytometry and histology analysis. RNA sequencing in vitro and after treatment in vivo gave us insight on expression of key immune molecules and allowed prediction of neoepitope expression on tumor cells. Bioluminescence allowed us to follow tumor growth in vivo and treatment duration and combination sequence was thoroughly studied. Use of immunodeficient Recombination activating gene 1 (RAG1) knocked out (KO) mice were used to assess T/B cell role in therapy efficacy, and we also addressed potential immunogenic cell death (ICD) induction in vitro.
RESULTS. SB28 has a lower mutational load than GL261 and a less immunogenic phenotype, with no constitutive Major histocompatibility complex I (MHC-I) expression.
Consistent with clinical data, SB28 was resistant to ICB with anti-PD-1 and anti-CTLA-4, whereas GL261 was highly sensitive with more than 50% of mice cured. SB28 was also resistant to temozolomide (TMZ), the standard care chemotherapy for GBM. On the other hand, SB28 was sensitive to treatment with a novel microtubule targeting agent (MTA), with only limited toxicity on immune populations. Nevertheless, MTA treatment did not sensitize to ICB therapy. There was no significant increase of neoepitopes after MTA treatment, but in vivo growth showed an Interferon gamma (IFNγ) signature on tumor cells by RNA sequencing. Flow cytometry analysis of brain infiltrating leukocytes (BILs) showed therapy-dependent modulation of the immune infiltrate. Notably, we revealed a synergistic effect of MTA and agonistic anti-CD40 treatment, which was conserved in RAG1 deficient mice and therefore independent of adaptive immune responses.
CONCLUSIONS. The SB28 mouse GBM model has a low mutational load comparable to most treatment-naïve human GBM, it is poorly infiltrated by immune cells and unresponsive to ICB. In contrast, the GL261 mouse GBM model is highly mutated, with a mutational load higher than most human cancers, it is well infiltrated and highly responsive to ICB. Survival of mice implanted with ICB resistant SB28 GBM cells could be extended by treatment with a combination of MTA chemotherapy and immunomodulation with agonistic anti-CD40
Ab, an effect that was T-cell independent. These results indicate that choice of in vivo models profoundly impacts decisions about suitability of treatment combinations for human GBM. We believe GBM heterogeneity and prior treatment history will impact therapy responsiveness in patients, and that pre-clinical testing in stringent models will be needed to tailor immunotherapeutic strategies for each tumor, thus empowering future clinical trials.
2. General introduction
a. Brain tumors
Brain tumors are categorized as primary or secondary tumors depending on their origin.
Secondary central nervous system (CNS) tumors are metastases from cancers outside the CNS and are all malignant. They account for the most frequent type of CNS tumors and originate from primary tumors principally of the lung, breast, skin, or kidney, in order of frequency[1].
Primary CNS tumors originate from the CNS itself, and can have different cellular origins.
Gliomas are tumors developing from the glial cells of the brain and are commonly classified as astrocytoma, oligodendroglioma, ependymoma, and oligoastrocytoma according to the World Health Organization (WHO)[2]. Further description is now recognized by the WHO classification based on presence of specific genomic characteristics such as isocitrate dehydrogenase (IDH) mutation and 1p19q co-deletion.
In parallel with such classification, gliomas are evaluated for their degree of aggressiveness and ranked with a grade. The most aggressive gliomas are grade IV, and called GBM. The combination of the histological and genomic characteristics associated with the grade gives the final classification of diffuse gliomas (Figure I). In this work, will focus mainly on GBM.
Figure I – Schematic representation of glioma classification adapted from[3]
WHO grade IV WHO grade III WHO grade II
Astrocytoma Oligodendroglioma
1p/19q non-codeleted
IDH mutant wildtype IDH
1p/19q codeleted
GBM is the most common primary brain tumor with an incidence of 7.23 cases for 100,000 patients a year, and a median age at diagnosis of 64 years[4]. We describe secondary GBM as tumors that transformed from lower grade to grade IV GBM, and are most of the time IDH mutated. Still, the vast majority are primary GBM that initially presents as grade IV.
Specific histological characteristics associated with GBM includes hypoxic necrotic foci, pseudopalisading necrosis and highly vascular stroma[5]. GBM is known to be one of the most vascularized tumors, and tumor associated vessels are mostly dysfunctional and thus cannot prevent development of hypoxic regions, another GBM feature[5]. In 2010, DNA sequencing of GBM from multiple patients identified different subtypes: proneural, classical and mesenchymal, based on presence of specific mutations (PDGFRA, IDH1, EGFR, and NF1) illustrating the inter-tumoral heterogeneity of GBM[6]. More recently, single cell analysis described the concurrent presence of all subtypes within the same tumor, illustrating the intra-tumoral heterogeneity and therefore limiting the clinical relevance of theses subtypes for patient classification and therapy prognosis stratification[7, 8].
Standard of care treatment for GBM consists of extensive surgical resection of the tumor followed by radio-chemotherapy. Radiotherapy is directed to the tumor location with a margin to encompass surrounding healthy tissue aiming at targeting the infiltrating cells.
TMZ is an alkylating agent used concomitantly with radiotherapy and maintained for 6 more cycles after radiation. One strong positive predictor of TMZ response is the methylation of the O6-alkylguanine DNA alkyltransferase (MGMT) promotor, if not present, the benefit of this chemotherapy is highly compromised[9]. Bevacizumab, an anti-VEGF (Vascular Endothelium Growth Factor) Ab prolongs Progression Free Survival (PFS) but not the Overall Survival (OS)[10, 11], and is not subjected to a consensus guideline, but rather follows country/institution specific use in Europe. Inevitably, GBM always recurs and Lomustine is one treatment option at this stage.
Overall the median survival for patients suffering from GBM is very low, with less than 10%
survival at 5 years[4], and the latest treatment improvement for GBM, based on adjunction of TMZ to radiotherapy dates from 2005[12]. Since then, no further therapy advances have been offered to patients with GBM, still, a growing number of clinical trials are evaluating combination strategies or novel compound to address this unmet need. Immunotherapy is of particular interest, given its remarkable efficacy in other cancer indications; this will be the primary focus of this thesis.
b. Animal models
Although in vitro experimentation allows precise study of cells, it cannot reproduce the complexity of an in vivo context. Moreover, the in vivo environment is more faithfully modeled when tumors are implanted orthotopically, rather than sub-cutaneously, which will not reproduce the particularities of the brain, critical for the study of GBM. In the context of translational studies where results observed in the model are aimed to be brought to the patient, differences in models can have major consequences and have to be considered, as will be shown in the results herein.
Xenograft models are the most used in cancer research. Established and well described cell lines, such as U251 or U87, initially derived from patients are common resources for in vitro studies and can be implanted in vivo in immunodeficient mice. Cells can also be harvested directly from patients and studied in mice as a personalized process called Patient Derived Xenografts (PDX). Although this approach allows a direct study of human cells in vivo, it requires the use of immunodeficient mice to avoid graft rejection, restricting its utility for immunotherapy studies. We will therefore focus on immunocompetent mouse models allowing the study of glioma immunotherapy strategies.
Spontaneous mouse models are generated by injection of vectors that will target a specific type of cell to deliver genetic alterations and lead to oncogenic transformation.
Genes targeted can be selected based on our knowledge of pathways implicated in human tumorigenesis adding an extra level of accuracy. If the spontaneous generation and precise genetic features can be reproduced with these strategies, incomplete tumor penetrance and difficulties at predicting the timeframe of tumor appearance are observed with some models[13].
Implanted mouse models consist of engrafting already transformed tumor cells.
Mechanisms to obtain tumor cells varies, but the most used is induction with carcinogen, and particularly methylcholanthrene. Multiple models derive from this technique, such as CT-2A, GL261 and GL26 models[13]. The strong reproducibility and predictability of the tumor kinetics gives a clear advantage in experimental settings even if engraftment is more artificial than spontaneous models.
Combining both approaches by implanting genetically manipulated cells adds a faithful genetic landscape to tumor growth predictability as it is the case for the SB28 model[14, 15].
Immunological considerations. Because discrepancies can exist between strains when studying precise immunological cell populations, the use of mice from the C57BL/6 (B6) strain is advantageous as it is the best immunologically described background. Syngeneic models in the B6 background are therefore favored for immunotherapy studies. Other tools are also available to better study the cancer-immune interaction with transfected variants of cell lines. For example, the GL261 model is syngeneic to the B6 background and multiple transfectants like ovalbumin (OVA)[16] are available. Such models offer the opportunity to pre-clinically study interactions between the immune system and tumor, even if in an artificial setting, and therefore better design treatment strategies. Because cells from the innate and adaptive immune system are described to interact with GBM in vivo, we need to better understand their specificities.
c. The immune system
i. Innate immunity
The innate immune system is the second level of protection against pathogens, after physical barriers such as the skin and before the adaptive immune system. Innate immunity is composed of different players, both cells and soluble factors; some of the key players are described below.
Macrophages are the first cells to encounter threats to tissues such as bacteria, viruses or cancer cells. Macrophages are resident cells in tissues, their primary action is to phagocytose microbes and debris and to initiate the inflammation process. They are established in tissues during embryogenesis, or derive from circulatory monocytes that differentiate toward macrophages once they leave the blood stream. In the brain microglial cells play the role of resident macrophages, but with some particularities, as we will see later. Macrophages recognize threats through pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) with specialized germ-line encoded molecules such as Toll-like receptors (TLR). A variety of TLRs exist with specific characteristics regarding their localization and ligands. Upon phagocytosis, macrophages will secrete inflammatory cytokines to further amplify the immune response.
Some of these cytokines as interleukin-1 (IL-1), IL-6 or tumor necrosis factor alpha (TNFα) will have systemic effects (inducing fever or liver production of acute phase proteins and complement), others, such as C-X-C motif chemokine 10 (CXCL10) will attract further leukocytes. As central cells involved in the immune response, macrophages are also involved in anti-tumor reactions, but they can have opposite effects such as directing effector cells to the threat, or on the contrary limiting their action, as it will be discussed
Interferon. Cells infected by viruses will secrete Type I Interferon (IFN). Type I IFN is composed of different types, the most well described being IFNα and IFNβ. They will have autocrine and paracrine effect on cells to interfere with viral replication and can also attract natural killer (NK) cells. IFN can impact on tumor cells and are described to be produced in the tumor microenvironment[18].
NK cells are lymphocytes from innate immunity lacking highly diverse antigen receptors like T and B lymphocytes, but express a variety of activating and inhibiting receptors. The balance of activation/inhibition signal will determine their functionality. The Natural Killer group 2D (NKG2D) receptor and cluster of differentiation 16 (CD16) are main activation molecules on NK cells, which are regulated by signaling through inhibitory receptors, including several of the killer-cell immunoglobulin-like receptors (KIR) receptors. Their activation by macrophages through IL-12 will lead to IFNγ production, a Type II IFN that will further activate macrophages. They have an intrinsic capacity at recognizing stressed cells, and particularly tumor cells[19]. With their role of macrophage support, interaction with dendritic cells (DC) and cytotoxicity, NK cells bridge innate and adaptive immunity and will be joined by T cells while the inflammation process continues.
ii. Adaptive immunity
Although innate immunity is activated with little delay against a new threat to the host, its specificity is limited to conserved structures on pathogens or stressed cells recognized through a defined and limited set of germline receptors. In contrast, adaptive immunity takes time to become functionally important, but displays an almost infinite, highly specific, recognition capacity and keep an immune memory of this response.
Lymphocytes are the major players of the adaptive immunity, encompassing B and T lymphocytes. B cells will become specialized for Ab production after their activation, and target pathogens in the circulation, in tissues, at mucosal sites, and soluble factors like toxins; whereas T lymphocytes are more involved in cellular immunity. While detection of Ab against tumor cells is described, their functional role is still unclear, particularly for solid tumors[20]. Therefore, in the next paragraphs we will focus on T cells, as their implication in tumor recognition and elimination has been widely recognized[20].
Receptors. B and T cells recognize their target – which is called the antigen (Ag) – through their B-cell receptor (BCR) and T-cell receptor (TCR), respectively. BCR and TCR structure differs, but they both possess constant and variable regions. Parts of the variable region are in contact with the Ag, and recognize only a specific part of the Ag, called the epitope. Consequently, multiple epitopes for the same Ag can exist. BCR are composed
of a heavy and light chain forming a Y shape. This receptor can be secreted and become an Ab when the B cells differentiate to a plasma cell, as opposed to the TCR, mainly formed by an α and β chain, which will never be secreted. Each lymphocyte has a specific receptor generated randomly, which can potentially bind to a precise epitope, which is an amino-acid sequence in the case of the TCR. This system is supported by somatic recombination, unique for B and T cells. The RAG protein complex will proceed to nearly random rearrangement of multiple genes segments (V, D and J) to generate each time a different sequence. This process is further completed by junctional diversity where random nucleotides are added to join these segments leading to an almost infinite generation of specific TCR or BCR sequences. The scope of all possible TCR generation possibilities constitutes the T cell repertoire of Ag recognition, unique for each individual, similar processes also lead to a diverse B cell repertoire.
Recognition. While the BCR can directly bind its Ag, the TCR needs a specific process to recognize the Ag. The Ag has to be presented as a peptide on a specific molecule called the major histocompatibility complex (MHC) and the TCR recognizes a so-called peptide:MHC complex. MHC are encoded by human leukocyte antigen genes (HLA) in humans and by the histocompatibility-2 (H2) genes in mice. These genes are known to be highly polymorphic, limiting the probability that 2 individuals have the same MHC allotype.
This has consequences, as MHC chemical structure will preferentially bind different types of peptides, meaning that not all peptides can bind a specific MHC and that 2 different individuals will not bind exactly the same pool of peptides. It also implies that a T cell specific for one Ag will not be able to recognize this Ag if it is presented on a different MHC allele; this concept is called MHC restriction.
MHC molecules are divided in two families which have different functions. MHC-I is constitutively expressed at the surface of healthy cells (except for red blood cells) and presents peptides from cytosolic proteins. In the cytosol, proteins are constantly degraded in peptides and presented on MHC-I as a sample of all cytosolic protein content. If a viral infection occurs, viral proteins will be presented, and T cells will recognize the cell as infected by a virus. Cancer cells will also display samples of their cytosolic proteins pool that can be recognized by immune cells, although this does not always happen efficiently.
MHC-II will present peptides from the extracellular compartment after phagocytosis. An exception occurs when a phagocytosed protein is expressed on MHC-I, this process is called cross-presentation.
T lymphocytes. While MHC molecules are specialized in the compartment they sample,
only by CD8 T cells, which can differentiate into cytotoxic T cells (CTL) and can destroy infected or cancerous cells. CD4 T cells need interaction with peptide:MHC-II and will have a more indirect effect by activating macrophages and B cells for a better elimination of extracellular pathogens, by phagocytosis and/or Ab production. CD4 T cells can also help CD8 T cells response through cytokine release or antigen presenting cells (APC) activation.
Antigen presentation. The normal process of adaptive immunity activation takes place in secondary lymphoid organs such as lymph nodes (LN). The Ag is either captured and brought to secondary lymphoid organs by professional APC, or can directly drain to secondary lymphoid organs and be captured by APC. DC are the most specialized APC, they are present in LN and also in tissues where they have a role of sentinel. To enter LN, naïve T cells have on their surface adhesion molecules specific for receptors expressed on high endothelial venules (HEV), the route to enter LN from the blood stream. A stronger interaction from adhesion molecules immobilizes the T cell that will cross the endothelium to enter the LN. This process is called diapedesis.
T cell activation. After adhesion with DC and recognition of the peptide:MHC complex, leading to intracellular signaling (signal 1), T cells need co-stimulation to become fully activated (signal 2). CD28 expressed on T cell will interact with B7.1 (CD80) or B7.2 (CD86) on the DC to provide this co-stimulation. Additional signaling, adhesion molecule clustering and polarization to one location of the cell will create a confined interface between the T cell and the APC, constituting the immunological synapse. The activation signal is transmitted intracellularly by the TCR complex, composed of the TCR and the CD3 molecule with multiple subunits, and specifically the ζ chain that activates transcriptional activator Nuclear factor of activated T cell (NFAT) to induce gene expression modification of the newly activated T cell. Activated T cells can then produce IL-2 which will have an autocrine effect and lead to clonal expansion of the specific T cell.
Tolerance to self Ag is essential for immune responses to avoid autoimmunity. Because TCR are randomly generated, some of them will be auto-reactive. During T cell development in the thymus, most self-reacting lymphocytes are eliminated, this process in known as central tolerance. Peripheral tolerance complements the process with the need of co- stimulation: absence of such signal 2 for the T cell will lead to anergy, a state of inactivation. Regulatory T cells (Tregs) also contributes to peripheral tolerance by inhibiting T cells partly through secretion of transforming growth factor-β (TGFβ) or IL-10.
While the random generation of TCR allows production of auto-reactive receptors, it ensures the possibility to recognize virtually any target (within the limitation of one’s MHC
allotype), such as newly arising pathogens, but also tumor specific Ag (TSA) that derive from mutated proteins in tumor cells, also call neoantigens (NeoAg). TSA are specific for tumors cells as opposed to tumor associated Ag (TAA) that are self-proteins over-expressed or aberrantly expressed on tumor cells. As a result, T cells responding to TAA should be subjected to central tolerance, but some of them escape this process, especially if they are of low avidity, and are described to react to tumor cells[21].
CD4 T cells can have distinct functions. Depending on the signal received during activation, they will polarize towards different subsets including T helper 1 (Th1), Th2 or Th17.
Th1 CD4 T cells are dependent on IL-12 and will produce mainly IL-2 and IFNγ that will support macrophage functions and production of certain Ab isotypes by B lymphocytes.
Their role in anti-tumor immunity is supported by pre-clinical work with detection of tumor specific Th1 CD4 T cells in mouse models[22, 23]. IL-4 will induce Th2 CD4 T cells that produce primarily IL-4 and IL-5 and promote mast cell and eosinophil activation,
"alternative" macrophage activation, and B cell Ab production, particularly of the IgE isotype. Th17, generated in the presence of TGFβ and IL-6 have been linked to responses against infections, in graft-versus-host disease, and tumor specific Th17 have been described to eradicate tumor cells[24].
On the other hand, Tregs are also a subset of CD4 T cells characterized partly by the constitutive expression of the transcription factor forkhead box P3 (FoxP3)[25], but they fulfill immunosuppressive functions partly through secretion of inhibitory cytokines such as IL-10 and TGFβ, and are described at the tumor site[26, 27].
CD8 T cells once activated will produce cytokines, mainly IFNγ, they can differentiate to CTL and produce factor such as perforin, granzymes, and granulysin (only in humans) packed in lytic granules that will triggers apoptosis once targeted cells are recognized.
Fas mediated killing also induces apoptosis, but through direct cell-cell interaction. Once activated, the T cell phenotype will evolve by modulating expression of various molecules, especially immunoregulatory molecules as we will see in the next paragraph. As a consequence, phenotypic characterization of T cells can help us determine their activation status and their functionality. A lot of data implicates CD8 T cells in anti-tumor immunity for GBM, in models and also in patients [28].
Immunoregulatory molecules. All over the course of their activity T cells will interact with molecules presented by the cells they encounter, which can have either positive or negative consequences, and which will determine the amplitude and quality of the T cell
role of maintaining self-tolerance and tissue integrity during inflammation, but their expression can be inappropriate during tumor growth, limiting the anti-tumor immune response. Agonists of co-stimulatory pathways or blockade of inhibitory molecules, can reveal the potential of immune response against cancer. Inhibitory signals are commonly called immune checkpoints as they control T cell activity. The most described molecules are cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1).
CTLA-4 is a crucial immune checkpoint expressed principally on T cells[29, 30] and mainly on activated CD8 T cells[31] or Tregs[32], and has also been described on activated mouse NK cells [33]. Because the affinity of CTLA-4 is stronger than CD28 for the same ligands (CD80 and CD86), its expression will prevent CD28 ligation and the necessary co- stimulatory signals for T cell activation[34]. CTLA-4 can also act as an activation signal, as its ligation on Tregs will favor their immunosuppressive activity[35].
PD-1 can be expressed on a wider range of immune cells such as T and B cells, Tregs and NK cells. In physiological conditions, PD-1 expression on T cells is transitionally induced upon activation to avoid overactivation[36]. But as we will see in the next section, prolonged PD-1 expression will limit T cell functionality, corresponding to an exhausted phenotype, as described below. A high proportion of T cells at the tumor site are described to be PD-1 positive, therefore calling into question their functionality. Ligands for PD-1 are programmed cell death ligand 1 and 2 (PD-L1 and PD-L2). PD-L1 expression on healthy cells is induced by IFNγ as a mechanism of protection for tissue integrity in the course of inflammation[37], but also on tumor cells as a mechanism of defense, limiting anti-tumor immune response[38, 39]. Macrophages can also express PD-L1, leading to an immunosuppressive effect on T cells, and their presence is described in the context of tumors, such as GBM[40].
Many other molecules have been associated with inhibition of lymphocyte activity as shown in Figure II. Among them, LAG-3 (CD223) is described to inhibit CD8 activity, while enhancing Treg function when binding to its ligand MHC-II[41], and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) mainly inhibits Th1 response when binding galectin-9[42].
Exhaustion of T cells is induced by chronic exposure to Ag and is characterized by loss of functionality and loss of ability to proliferate. Persistent expression of PD-1[43] and co- expression of other immune checkpoint molecules like LAG-3[44] and TIM-3[45] have been described on exhausted T cells. This state was initially described in studies of chronic viral
infections, but also applies to anti-cancer immunity as persistence of tumor cells lead to chronic Ag exposure.
Memory. After resolution of inflammation and elimination of the pathogen, in the case of infection, the immune system keeps memory of the specific antigens encountered by the generation and maintenance of specialized memory T and B cells. These memory cells can be easily re-activated to efficiently and rapidly elicit a functional response upon pathogen re-encounter, or potentially provide long-term anti-tumor protection that is more effective than short-term effector cells[46].
Figure II – Immunoregulatory interactions adapted from[47]
iii. Brain particularities
More than 50 years ago, transplantation experiments showed that allogenic transplants were successfully engrafted in the brain as opposed to peripheral sites, the latter being rapidly rejected by the host[48]. For this reason, the CNS has long been considered as immune privileged, and the description of a blood brain barrier (BBB), lack of conventional lymphatics and absence of professional APCs contributed to maintain this concept[49].
Despite these features, immunological reactions can be observed in the brain, even if they are more controlled to preserve vital neurological function, as in the confined cranial space, uncontrolled inflammation would have more dramatic effect than anywhere else in the body. Hence, the postulate of immune-privilege has been now revised and the brain may not be privileged, but rather immune-specialized as we will see below.
The BBB is a highly specialized system to control influx and efflux of cells but also Ab, metabolites or chemicals between the vascular system and the brain parenchyma. It is composed of specialized endothelial cerebrovascular cells bound by tight junctions, that lack trans-endothelial fenestration, and have low pinocytic activity. Pericytes and astrocyte endfeet complete the barrier on the parenchymal side. This mechanism leads to tightly closed communications, nevertheless, factors such as TNF and nitric oxide (NO) can transiently open the barrier for DC and macrophages [50]. Activated T lymphocytes can also cross the BBB but expression of the integrin α4β1 is critical for their entry in the brain[51].
CNS lymphatics. If the BBB is not totally impermeable to immune cells, it has been argued that detection of Ag would be impaired in the CNS because of the lack of DCs and classical lymphatics. This has now evolved with the recent discovery that a meningeal lymphatic system exists and can drain Ag to the deep cervical LN for T cell activation[52], but this finding has to be confirmed to exist also in humans and to be applicable to all brain compartments, specifically the parenchyma where GBM develops.
Microglia are the most important population of myeloid cells in the healthy brain as classical macrophages are poorly represented under physiological conditions. Microglia have an embryonic progenitor origin and their persistence is due to self-renewal rather than replenishment from circulating bone marrow progenitor[53]. These cells endorse the role of resident macrophages of the CNS by actively surveilling the brain parenchyma, detecting threats and migrating to damaged sites. They are activated upon signals like ATP and can acquire more motility as well as phagocytosis[54]. Their role as APC has been described, even if they are less potent than classical professional APCs[55]. Still we can
consider microglia as the sentinel of the brain parenchyma, a role also fulfilled by the few macrophages present in the CNS, mostly in perivascular areas[49].
d. Immuno-Tumoral interactions
i. Cancer-immunity cycle
The idealized cancer-immunity cycle[56] is summarized in Figure III, where all the steps of immune response mentioned in the previous sections are adapted to anti-tumor immunity.
The 7 steps focus on T cell mediated tumor killing but other immune cells influence T cell efficacy and have to be considered. Moreover, every phase is highly regulated, involving different players; immune checkpoints or agonistic signals are involved all the way until tumor cell killing. We will see how the tumor-immune system interactions evolve, what mechanisms are interfering with the optimal course of this cycle, and ultimately how can we impact on these factors to make this cycle flow.
Figure III – The Cancer-Immunity cycle, adapted from[56]
ii. Immunoediting
The concept of immuno-surveillance was initially postulated at the beginning of the 20th century but formally hypothesized by Burnet & Thomas in the 1950s[57, 58]. At that time the scientific community was skeptical and it took years to convince people through finer description of the concept while better experimental tools were developed. Follow-up studies of transplanted patients confirmed an increase of overall cancer incidence with immunosuppression, including a few cases of gliomas[59]. The concept really matured in the last 20 years when Dunn, Schreiber and colleagues developed the concept from immuno-surveillance to immunoediting[60]. The immunoediting concept relies on the 3 Es:
Elimination, Equilibrium, Escape.
Elimination. The first step for elimination of cancer cells is their recognition by the immune system. Because the immune system has evolved for years toward optimal detection for pathogens, it is not primarily designed to tackle tumor cells, but this step is central.
Development of a tumor in the tissue will cause damage and induce inflammation after activation of the innate immune system. Lymphocytes can also recognize tumor cells based on their Ag signature through TSA and/or TAA and further amplify the anti-tumor response, leading to elimination of some or all of the tumor cells.
Equilibrium. Recognition of the tumor cells and induction of their elimination will lead to the second E – equilibrium – where a Darwinian process of selection is imposed to the tumor. Under the immune system pressure, cancer cells will be sculpted toward a less immunogenic profile. For example, loss of MHC-I expression is one illustration of immunoediting observed in patients[61]. If the immune system adapts its response to the less immunogenic tumor cells through epitope spreading, the process will stay at equilibrium with constant evolution of tumor cells and adaptation of the immune response.
Epitope spreading is a concept when an effective specific T cell response toward a defined epitope will favor the activation of other T cell clones specific for other epitopes through induction of a favorable environment and therefore broadening the immune response.
Escape. Inevitably some tumor cells will escape, depicting the 3rd E of the concept and lead to progression of the tumor. Tumors can escape immune recognition and elimination by multiple means that we will discuss in the next point.
iii. Glioma escape
As we have seen above, if we are confronted with progressive cancer growth, it is because ultimately tumors escape immune control and lead to tumor progression.
Different strategies can lead to tumor escape, and we will study major pathways already described for GBM. First, as stated before, the sole location of GBM in the CNS makes it more difficult to be efficiently eliminated by the immune system. Second, patients with GBM have a general immunosuppressed state with low CD4/Treg ratio[62] worsen by the radio/chemotherapy treatment and use of steroids [63]. GBM also exerts a broad panel of strategies to evade immune control as we will see below.
The tumor microenvironment (TME) of GBM is not in favor of immune activity, with only sparse T cell infiltration[64] and presence of Tregs[65-67]. It has sometimes been shown that infiltration of Tregs correlates with higher grade and progression of the tumor but its negative prognostic factor is controversial[68].
Myeloid cells. In GBM, 30 to 50% of tumor mass can be composed of myeloid cells and their abundance also correlates with grade[69]. Discriminating activated microglia and macrophages is not an easy task, but markers such as CD49d has been suggested to segregate these two populations[70]. GBM is described as a tumor highly infiltrated by tumor associated macrophages (TAM) that can have opposite profiles with a continuous spectrum going from the anti-tumoral M1 phenotype to the pro-tumoral M2 phenotype, the latter being particularly highly represented in GBM[71]. If CD163 and CD204 have been associated with M2 macrophages phenotype, the final conclusion of their status has to be tested functionally, like secretion of known M2 associated immunosuppressive factors such as TGFβ, VEGF, or matrix metalloproteinase (MMP)-2 and MMP-9[72].
Myeloid derived suppressor cells (MDSCs) are another immunosuppressive subtype of cells and their presence correlates with less T cell infiltration in human and mouse GBM[73, 74].
Hypoxia has been described to attract this immature myeloid subpopulation[75] which exerts immunosuppressive functions partly through galectin-1 which negatively regulates T cell survival and promotes accumulation of Tregs in the TME[76].
Soluble and Cell mediated Factors. Glioma cells actively impact the immune cells by secreting TGFβ known to inhibit T cells[77, 78], induce Tregs[79], and acts on TAM favoring their M2 phenotype[80]. As both Tregs and M2 TAM can secrete TGFβ and IL-10, this creates an autocrine and paracrine loop that amplifies the immunosuppressive state of the TME.
GBM can also protect itself by expressing molecules like PD-L1 on its surface to avoid T cell activity, acting like a “molecular shield” to CTL[40, 81]. In this regard, the common phosphatase and tensin homolog (PTEN) gene loss observed in GBM is associated with overexpression of PD-L1[82]. Additionally, down regulation of MHC-I leads to less immune
in the TME can also affect T cell activity, such as production of lactate by tumor cells which inhibits T cell function[84]. Expression of indoleamine 2,3-dioxygenase (IDO)[85] and arginase[86] by tumor cells or MDSCs will starve T cells from tryptophan and arginine, respectively, which is detrimental to the immune response as these two amino acids have been described to favor T cell survival and anti-tumor immunity[87, 88].
iv. Immune context
With regard to all mechanisms adopted by GBM to limit immune reactivity and T cell infiltration, we can consider this tumor as immunologically “cold”. A concept introduced by Galon et al. in 2006 [89] classifying the tumors according to their immune contexture [90, 91]. According to this concept, a cold tumor is described as poorly infiltrated with effector immune cells leading to a dominant immunosuppressive TME, compared to hot tumors highly infiltrated with immune favorable TME. Melanoma would be the best example of a “hot” tumor, but each patient has a distinct tumor with a specific TME immune signature[92].
e. Immunotherapy for cancer
Based on the growing knowledge of immune-cancer interactions, and understanding of the different defense mechanisms, multiple strategies aiming at revealing the immune system efficacy against tumors are now developed. But immunotherapy has been used for years, with Bacillus Calmette-Guérin (BCG) instillation in the case of bladder cancer, to activate local immunity, first described in 1976[93]. The principle of the graft versus tumor reaction observed and expected after allogenic stem cell transplantation is also a form of immunotherapy, and IL-2 therapy for patient with melanoma and renal cancer was already by the Food and Drug Administration (FDA) approved in the 1990s. Still, the real burst of immunotherapy drug approval and use in the clinic started in the last 10 years and is now exponentially increasing.
Different methods exist to activate the anti-tumor immune reaction; they are traditionally divided in two categories with active immunotherapy being vaccination, and passive immunotherapies composed of different means to activate the immune system by administration of immune component like Ab. Nowadays this differentiation seems outdated as even passive immunotherapies requires active collaboration of the host immune system to be efficient. Still, different approaches can be taken and some of them will be described in this section.
i. Vaccination
Prophylactic vaccination targets viruses known to drive oncogenic transformation of infected cells and tumor development. By preventing infection of such viruses, prophylactic vaccination prevents potential cancer development. One particular example is vaccination against Human papillomavirus (HPV), known to induce cervix cancer, or hepatitis B virus (HBV) where chronic infection can lead to hepatocellular carcinomas. These prophylactic vaccinations are available in most countries and have already proven their efficacy by decreasing the incidence of their respective cancers[94, 95]. But because the majority of cancer has no known viral origin, this strategy is limited.
Therapeutic vaccination aims at helping the host immune system to recognize and efficiently initiate anti-tumor immune response against an already developed tumor. To do so, the immune system needs to be educated at recognizing specific Ag. Depending on the technique used to induce vaccination, either TAA, TSA, or both can be recognized.
Whole-tumor cell vaccination will take both categories of Ag into account but vaccine based on tumor Ag, tumor peptide or DC pulsed with defined Ag requires a selection of the Ag prior to vaccination.
TAA can be identified by comparing expressed molecules of tumor cells to healthy cells by RNA sequencing or surface staining, but identification of TSA can be more complicated. TSA derives from mutations that can be sequenced and defined. If this mutation if expressed, the different peptides containing the mutation can be predicted.
Then based on the specific sequence of the peptide, specialized algorithms can predict its binding to MHC-I based on the chemical affinity of the peptide and the precise MHC allotype of the individual. This can predict presentation of the peptide harboring the mutation on MHC molecules. Elution of the peptides presented at the surface of the tumor cells is another strategy to identify presented peptides.
While the Ag gives signal 1 to the T cells, they also need signal 2 provided by APC for full activation. To induce this co-stimulation, Ag needs to be administered with an adjuvant aiming at activating the APC. It has even more importance in the case of anti-cancer vaccination as the immune system already failed to efficiently recognize the tumor cells and potential peripheral tolerance has been induced.
There is currently only one FDA approved therapeutic vaccination, Sipuleucel-T, a DC- based vaccine for a specific prostate cancer approved in 2010. For GBM, different approaches have already been evaluated. A peptide vaccine for EGFRvIII has been
receptor, but had to stop prematurely because of lack of efficacy[96]. The IMA-950 trial tested a multipeptide vaccine, and could induce immune responses to some of the peptides[97, 98]; other approaches with whole tumor lysate, or DC based vaccines have also been conducted[99-101]. Even if these results are encouraging and provide the scientific rationale to further develop GBM cancer vaccines, we still await phase III results to reach definitive conclusion about their clinical efficacy.
ii. Adoptive cellular therapies
Adoptive cellular therapies (ACT) are based on the transfusion of autologous T cells specific for the tumor cells. They can be selected for tumor specificity from a pool of patient’s derived lymphocytes or engineered to acquire tumor specificity.
Tumor infiltrating lymphocyte (TIL) therapy aims at identifying, selecting, activating and expanding tumor specific T cells in vitro before re-infusing them into the patient as a
“boosted” population of autologous T cells. Expansion of tumor specific T cells have been achieved for some melanoma patients but has not beed approved for clinical use yet[102]. Because tumor specific T cells cannot always be isolated from patients, cell engineering can address this issue.
TCR engineering allows to force the expression of a selected TCR at the T cell surface [103].
TCR transduction can convert a T cell of irrelevant specificity to a tumor specific T cell by incorporating a new TCR, bypassing the need for natural generation of tumor specific T cells. Virtually all Ag could be targeted with this strategy, but is still dependent on MHC presentation. TCR therapy can be developed to target known mutated peptides as it has been recently demonstrated for diffuse intrinsic pontine glioma in a mouse model[104].
Chimeric antigen receptor (CAR) T cells are engineered T lymphocytes that expresses an artificial receptor on their surface. This receptor is made of the variable fragment of an Ab fused to the intracellular signaling pathway of the TCR and various co-stimulatory domains.
This allows the activation of the engineered T cell when in contact with a selected Ag, and avoids the limitation of MHC restriction. However, the approach is only applicable to cell suface expressed antigens. Tisagenlecleucel was the first CAR therapy approved by the FDA. It targets CD19 on B cells and is now indicated for some cases of B cell acute lymphoblastic leukemia and large B cell lymphoma[105]. If this strategy of immunotherapy is highly promising, the only two CAR therapies approved to date are targeting the same antigen, CD19, illustrating the difficulty of identifying targets homogeneously expressed by all malignant cells in a patient or across different patients, a key limiting factor for CAR T cells.
For GBM, work is underway to develop CAR T cell therapy, especially targeting EGFRvIII;
these therapeutic cells have been proven to be safe, and they efficiently reach the tumor site[106]. But as in other solid tumor, CAR monotherapy seems to lack efficacy, potentially because its final potency relies in inducing epitope spreading, and therefore would benefit from combination with ICB e.g., a strategy currently being evaluated.
iii. Therapies using antibodies
Antibodies can specifically target tumor cells, usually TAA but could also target TSA. They can induce Ab-dependent cell-mediated phagocytosis (ADCP) by opsonizing the tumor cells, or target receptors involved in tumor cell survival. Antibodies can also be bound to drugs conjugates, allowing a more tumor-specific drug distribution. But downsides exist for targeted Ab approach with potential off-tumor side effects (especially with TAA) or acquired resistance of tumor cells no targeted by the Ab. Moreover, specifically for GBM, Ab access to the brain is one strong limitation of this approach [107]. Ab can also interfere with processes required for optimal tumor growth, like bevacizumab, targeting VEGF that will interfere with angiogenesis. Other Ab targeting approaches are approved and used in the clinic; Rituximab targets CD20 on B cells for the treatment of non-Hodgkin lymphomas, and Trastuzumab targets the HER2/neu oncoprotein on some breast cancer cells. For GBM, Ab against EGFRvIII[108] and also drug-conjugated have been developed and are currently under investigation in clinical trials.
iv. Immunomodulatory antibodies
All the above-mentioned immunotherapeutic strategies rely on the presence, or identification and sometimes selection of tumor Ag, this raises technical challenges and can also lead to immunoediting and eventually immune escape. Immunomodulatory Ab target specific molecules involved in immune interaction to either “release the breaks” or
“press the gas pedal”[90] on the immune system in general to allow a better activation state for effective anti-tumor immunity, without the limitation of identifying tumor specific targets.
Immune checkpoint blockade, as already discussed, consists of blocking inhibitory signals to the T cell, allowing an overall more activated state of the lymphocyte[47]. Multiple targets exist, as all steps of the cancer-immunity cycle can be targeted (Figure III). CTLA- 4, was first ICB target described by Allison and colleagues[109], the PD-1/PD-L1 axis is another well described ICB target, but others like TIM-3 or LAG-3 are also associated with enhanced tumor-immunity when blocked[56]. For GBM, evidence for efficacy of blocking PD-1, PD-L1, CTLA-4 and TIM-3 exists, but mainly for one model: GL261[110-114].
In clinical practice, CTLA-4, PD-1 and PD-L1 blocking Ab are already FDA approved and the scope of their applications is increasing very fast, leading a revolution in cancer treatment. The unexpected clinical results achieved with ICB for some cancer indications drew enthusiasm for all cancers and for GBM a myriad of clinical trials are currently evaluating the efficacy of ICB (as reviewed in[115]). The first preliminary result evaluating anti-PD-1 monotherapy for recurrent GBM was negative[116], but a more recent study showed efficacy of anti-PD-1 therapy when administered in a neoadjuvant setting for recurrent GBM, compared to adjuvant only [117]. Still, we will need to wait for more data from the ongoing clinical trials to reach definitive conclusions about GBM sensitivity to ICB, especially for primary GBM.
Agonistic Ab. By activating pathways necessary for an effective anti-tumor response, agonistic Ab can potentially compensate for the lack of spontaneous stimulation needed for tumor rejection. Anti-CD40 is a prototypic example as it targets DC and macrophages that can then further activate other cell subtypes. Efficacy of agonistic anti-CD40 therapy for GL261[118, 119] and SB28[14] is already described, and clinical trials in pediatric CNS tumors are currently being conducted. OX40 or Inducible T-cell COStimulator (ICOS) expressed on T cells would be other potential targets for agonistic Ab to further activate T cells, as well as CD137 for which pre-clinical data supports efficacy in GL261[120]. Although agonistic Ab therapies are evaluated in clinical trials no data is available for GBM yet.
Biomarkers identification is an active field of study for immunotherapy, because if the therapeutic effect can be impressive, they account only for a subset of patients, and up to now, no clear predicting factor has been identified. For many tumors, PD-L1 expression level is correlated with response to ICB targeting PD-1/PD-L1 pathway, but the evaluated thresholds are inconsistent, ranging from >1% to >50%[121]. Furthermore, many cases of patients with no PD-L1 signal detected benefited from ICB targeting PD-1/PD-L1 axis, arguing for the poor predictive value of PD-L1 staining on its own[122]. In the case of GBM, PD-L1 can be detected in patients, but with a very wide range depending on the technique of detection[40, 81]. Because PD-L1 is not exclusively expressed on tumor cells, but also macrophages[122], high levels of PD-L1 signal could be a sign of a highly immuno- suppressive TME that monotherapy targeting PD-1/PD-L1 pathway will not sufficiently overcome; and this situation would rather need a combination of therapies.
Correlation of better response to ICB with a higher mutational load has also been observed multiple times, and the rationale lies in the higher generation of neoantigens[123]. GBM is predicted to only occasionally generate neoepitopes, therefore hampering a straightforward application of current immunotherapies aiming to target TSA, but not
precluding them[123]. Patients with mismatch repair deficient tumors are also generally better responders to ICB regardless of the tumor type, because of the high number of mutations, and anti-PD-1 therapy has been recently clinically approved for patients with micro-satellite instability (MSI), regardless of the histology. This has also been illustrated for GBM with reported cases of hypermutated GBM responding to ICB[124].
Despite numerous parameters actively studied, no clear individual predictive marker for immunotherapy responsiveness has been identified so far. Because of the complexity of tumor-immune interactions, predictive scores are being developed that incorporate multiple parameters to assess immune context of a tumor, such as the immunogram, but this still needs validation[125].
Side effects are common with immunomodulatory Ab. All ICB molecules block physiological pathways of the immune system aiming at controlling the reaction by avoiding overactivation of T cells. Because they do not target tumor specific T cells but instead interfere with the global mechanisms of activation control, the most frequent side- effect is auto-immunity. In the case of agonistic Ab, the problem is even more pronounced as they non-specifically activate the immune system. Combining multiple ICB pathways also increase side effects, as described for GBM, where more than 50% of patients had to discontinue a combination strategy in a recent clinical trial[126]. Nevertheless, as clinical practice has now more experience with immunotherapy, better management and anticipation of potential side effects is assured.
v. Other immunotherapeutic approaches
Oncolytic viruses were initially developed to infect and kill tumor cells specifically, but it has been also clearly shown that they can lead to immune stimulation[127]. T-vec, an oncolytic approach to treat melanoma is already approved since 2015[128]. Pre-clinical efficacy of such strategy for GBM has already led to development of clinical trials of oncolytic viral therapy[129, 130] and results are awaited to judge their clinical efficacy.
Metabolic modulators can impact on the TME to favor an immuno-supportive context. IDO inhibitors will inhibit tryptophan degradation, promoting growth and survival of T cells. This strategy is actively being explored and pre-clinical data supports its effect on GL261[112, 114]. Unfortunately, recent data from clinical trials could not reveal any effect of IDO inhibitors either alone or in combination with an anti-PD-1 Ab for solid tumors, leading to interruption of many clinical trials[131]. Comprehension of the reasons of failure will help to improve IDO therapy as monotherapy or in combination.
Because IDH mutation leads to generation of an immunosuppressive metabolite, inhibition of this process is explored for GBM and proved efficacious in pre-clinical models[132];
further investigation could lead to clinical trial investigation.
vi. Immunotherapeutic combinations
Most of the approved immunotherapeutic treatments are targeting non-redundant pathways and could therefore be rationally combined together. If the myriad of other options currently being investigated are taken into consideration, the total number of possible combinations would be too high to allow complete evaluation. However, strategies based on clinical data, such as the description of GBM TILs co-expressing PD-1 and TIM-3[111], can help us tailor better combinations and test them pre-clinically in carefully selected models. Stimulating different arms of the immune system is another rational approach, especially for GBM that is particularly heterogeneous in its immune composition, with few T cells and more myeloid cells, specially M2 macrophages[71].
Combination of new immunotherapies can be usefully explored, or their combination with current standard of care treatments as discussed below.
Chemotherapy can potentiate immunotherapy by different means. By killing tumor cells, chemotherapy will induce Ag release and favor APC presentation of those Ag, potentially leading to anti-tumor immunity. But chemotherapeutic agents do not induce cell death equally, some induce more expression of DAMPs that better activate the immune system.
This concept is called immunogenic cell death (ICD), and anthracyclines, oxaliplatin or bortezomib are well described ICD inducers[133, 134]. For GBM, the standard of care TMZ is known to negatively impact on the immune system and patients may be more susceptible to infections during treatment[135]. Furthermore, mouse models treated with high dose of TMZ has been described to prevent PD-1 efficacy[136]. Nevertheless, TMZ is also proposed to decrease the number of Tregs in GBM[137]. In this regard, careful combination of TMZ and immunotherapy needs to be further explored.
Radiotherapy will induce cell death and Ag release and is another well described ICD inducer. It has been hypothesized to improve ICB therapy by triggering a Type I IFN response, inducing MHC-I expression and generating NeoAg and uptake by APCs while decreasing MDSC presence in the TME[138]. However, radiotherapy can induce TGFβ and is associated with lymphodepletion, depending on the mode of delivery[138].
Nonetheless, the overall effect of radiation seems beneficial in combination with immunotherapy in the GL261 model, as increased CD8 infiltration, with an activated phenotype is observed with anti-PD-1 combination[139]. Clinical trials are actively studying
radiotherapy and immunotherapy combination for GBM patients, also supported by evidence of efficacy in other solid tumor indications.
Overall, combination strategies seem to be a rational way forward as monotherapy may have only limited efficacy, especially for GBM. But rational combination protocols are needed, as the growing number of possible associations of treatment is impossible to fully assess. To address this point, Table I assembles all published pre-clinical data evaluating ICB as monotherapy and in combination in syngeneic mouse models of GBM.
Model Therapy/Target Effect Group/year Ref GL261
MCA 4-1BB Efficacy of therapy Shu 2001 [140]
CT-2A PD-1 CTLA-4
Oncolytic viruses
Efficacy of some
combinations Rabkin 2017 [141]
CT-2A PD-1 CTLA-4
Oncolytic viruses
Efficacy of triple
combination only when injected IC
Rabkin 2017 [142]
GL261 PD-1 CTLA-4
Efficacy of monotherapy and combination
Reardon 2016 [110]
GL261 GL261- IDO1-KO
IDO PD-1
Radiotherapy IDO KO mice
Multiple treatments
efficacious depending on mice and cells
Wainwright 2018
[112]
GL261 GL261- IDO1-KO
CTLA-4 PD-L1
IDO KO mice
Efficacy of combination in
wild-type mice Wainwright
2017 [113]
GL261 GITR Only if intra tumoral injection Lesniak 2016 [143]
GL261 GL261- IDO1-KO
CTLA-4 PD-L1 TMZ
IDO inhibitors IDO KO mice
Multiple treatments
efficacious depending on mice and cells
Lesniak 2014 [114]
GL261 OX40 Vaccine
(Irradiated GL261 + GM-CSF)
Efficacy of monotherapy
and combination Curry 2018 [144]
GL261 CTLA-4 Vaccine
(Irradiated GL261 + GM-CSF)
Efficacy of monotherapy and combination
Curry 2012 [145]
GL261 PD-1 CTLA-4 IDO inhibitors IDO KO mice
Efficacy of only PD-1 Agostinis 2017 [146]
GL261 PD-1
TMZ Efficacy only with
combination Zhang 2018 [147]
GL261 PD-1 OX40
Vaccine (GL261 secreting GM-CSF)
Efficacy of combinations or
PD-1 in monotherapy Curry 2019 [148]
GL261 luc- mCherry
CTLA-4
Dexamethasone Efficacy only with
combination Gilbert 2018 [149]
GL261 PD-1
Oncolytic virus Efficacy only with
combination Galanis 2017 [150]
GL261 PD-1 Efficacy of monotherapy De
Vleeschouwer 2017
[151]
GL261 CD137 No efficacy Lim 2011 [120]
Model Therapy/Target Effect Group/year Ref GL261-luc PD-1
TIM-3
Radiotherapy
Efficacy of monotherapy for PD-1 and radiotherapy, and efficacy of all combinations.
Combination of 3 with best efficacy
Lim 2017 [111]
GL261-luc PD-1
IL-15 superagonist Radiotherapy
Efficacy of monotherapy and combinations except for radiotherapy
Lim 2016 [152]
GL261-luc 4-1BB CTLA-4
Radiotherapy
Efficacy of radiotherapy with CTLA-4, CTLA-4 with 4-1BBB and triple combination
Lim 2014 [153]
GL261-luc PD-1
Radiotherapy Efficacy only with
combination Lim 2013 [139]
GL261-luc GITR
Radiotherapy
Efficacy of monotherapy and combination
Lim 2016 [154]
GL261-luc PD-1 TMZ
Carmustine
Efficacy of monotherapy of Carmustine or PD-1 and their combinations. Local
temozolomide with PD-1
Lim 2016 [136]
GL261-
luc2 PD-1
Gene-mediated cytotoxicity
Efficacy of monotherapy
and combination Lawler 2018 [155]
GL261- Quad SB28
CD40 Celecoxib
Efficacy only with combination
Okada 2014 [14]
NSCL61 GL261 bRiTs-G3
CD40 Efficacy for NSCL61and
bRiTs-G3 by injecting locally
Tominaga 2016
[119]
NSCL61
GL261 CD40 Ox40
Vaccination (irradiated cells)
Efficacy of all monotherapy
and all combinations. Tominaga
2015 [118]
SMA-560 CTLA-4 Efficacy Sampson 2007 [156]
SB28
GL261 CTLA-4 PD-1 CD40 BAL101553
Efficacy of BAL101553 alone and synergy with CD40 stimulation
Efficacy of PD-1 and CTLA-4 inhibition for GL261, and only combination of BAL101553 and CD40 stimulation
Walker 2018
And this thesis [15]
Table I – ICB strategies and combinations in syngeneic mouse models of GBM.
3. Objectives
As discussed, the interactions between tumor cells and the immune system are complex, with an additional level of complexity in the context of GBM in which all the particularities of CNS immunity have to be considered. In this context advanced mouse models faithfully recapitulating key characteristics of human GBM are necessary to allow translation of the concepts studied in the models to patients. Even though all parameters involved in immunotherapy responses are fully elucidated, specific characteristics such as T cell tumor infiltration and mutational load are linked to success of ICB and need to be carefully reproduced in the models used to develop immunotherapy. Furthermore, we now know that despite the indisputable success of immunotherapy for some cancers, this is not the case for all tumors, and strategies to broaden efficacy of immunotherapy needs to be tailored to tumor characteristics.
In this context, we endeavor in this thesis to study the application of immunotherapy specifically for GBM, by addressing two primary aims:
1. To assess whether proposed prognostic indicators of responsiveness to PD-1/PD-L1 and CTLA-4 blockade can be extended to two syngeneic mouse GBM models.
2. To test whether alternative immunomodulators to ICB, and new-generation chemotherapies can be considered for categories of GBM that are ICB-resistant.
We pursued these aims through two complementary projects that are presented below as two separate chapters. The first one corresponds to a published paper and the second one is presented as a manuscript that will be submitted.
