Combination of vasculature targeting, hypofractionated radiotherapy, and immune checkpoint inhibitor elicits potent antitumor immune response and blocks tumor progression.

BACKGROUND: Tumor endothelial marker 1 (TEM1) is a protein expressed in the tumor-associated endothelium and/or stroma of various types of cancer. We previously demonstrated that immunization with a plasmid-DNA vaccine targeting TEM1 reduced tumor progression in three murine cancer models. Radiation therapy (RT) is an established cancer modality used in more than 50% of patients with solid tumors. RT can induce tumor-associated vasculature injury, triggering immunogenic cell death and inhibition of the irradiated tumor and distant non-irradiated tumor growth (abscopal effect). Combination treatment of RT with TEM1 immunotherapy may complement and augment established immune checkpoint blockade. METHODS: Mice bearing bilateral subcutaneous CT26 colorectal or TC1 lung tumors were treated with a novel heterologous TEM1-based vaccine, in combination with RT, and anti-programmed death-ligand 1 (PD-L1) antibody or combinations of these therapies, tumor growth of irradiated and abscopal tumors was subsequently assessed. Analysis of tumor blood perfusion was evaluated by CD31 staining and Doppler ultrasound imaging. Immunophenotyping of peripheral and tumor-infiltrating immune cells as well as functional analysis was analyzed by flow cytometry, ELISpot assay and adoptive cell transfer (ACT) experiments. RESULTS: We demonstrate that addition of RT to heterologous TEM1 vaccination reduces progression of CT26 and TC1 irradiated and abscopal distant tumors as compared with either single treatment. Mechanistically, RT increased major histocompatibility complex class I molecule (MHCI) expression on endothelial cells and improved immune recognition of the endothelium by anti-TEM1 T cells with subsequent severe vascular damage as measured by reduced microvascular density and tumor blood perfusion. Heterologous TEM1 vaccine and RT combination therapy boosted tumor-associated antigen (TAA) cross-priming (ie, anti-gp70) and augmented programmed cell death protein 1 (PD-1)/PD-L1 signaling within CT26 tumor. Blocking the PD-1/PD-L1 axis in combination with dual therapy further increased the antitumor effect and gp70-specific immune responses. ACT experiments show that anti-gp70 T cells are required for the antitumor effects of the combination therapy. CONCLUSION: Our findings describe novel cooperative mechanisms between heterologous TEM1 vaccination and RT, highlighting the pivotal role that TAA cross-priming plays for an effective antitumor strategy. Furthermore, we provide rationale for using heterologous TEM1 vaccination and RT as an add-on to immune checkpoint blockade as triple combination therapy into early-phase clinical trials.

The dark side of tumor-associated endothelial cells.

Angiogenesis is a hallmark of cancer and a requisite that tumors must achieve to fulfill their metabolic needs of nutrients and oxygen. As a critical step in cancer progression, the 'angiogenic switch' allows tumor cells to survive and grow, and provides them access to vasculature resulting in metastatic progression and dissemination. Tumor-dependent triggering of the angiogenic switch has critical consequences on tumor progression which extends from an increased nutrient supply and relies instead on the ability of the tumor to hijack the host immune response for the generation of a local immunoprivileged microenvironment. Tumor angiogenic-mediated establishment of endothelial anergy is responsible for this process. However, tumor endothelium can also promote immune tolerance by unbalanced expression of co-stimulatory and co-inhibitory molecules and by releasing soluble factors that restrain T cell function and induce apoptosis. In this review, we discuss the molecular properties of the tumor endothelial barrier and endothelial anergy and discuss the main immunosuppressive mechanisms triggered by the tumor endothelium. Lastly, we describe the current anti-angiogenic therapeutic landscape and how targeting tumor angiogenesis can contribute to improve clinical benefits for patients.

Local endothelial complement activation reverses endothelial quiescence, enabling t-cell homing, and tumor control during t-cell immunotherapy.

Cancer immunotherapy relies upon the ability of T cells to infiltrate tumors. The endothelium constitutes a barrier between the tumor and effector T cells, and the ability to manipulate local vascular permeability could be translated into effective immunotherapy. Here, we show that in the context of adoptive T cell therapy, antitumor T cells, delivered at high enough doses, can overcome the endothelial barrier and infiltrate tumors, a process that requires local production of C3, complement activation on tumor endothelium and release of C5a. C5a, in turn, acts on endothelial cells promoting the upregulation of adhesion molecules and T-cell homing. Genetic deletion of C3 or the C5a receptor 1 (C5aR1), and pharmacological blockade of C5aR1, impaired the ability of T cells to overcome the endothelial barrier, infiltrate tumors, and control tumor progression in vivo, while genetic chimera mice demonstrated that C3 and C5aR1 expression by tumor stroma, and not leukocytes, governs T cell homing, acting on the local endothelium. In vitro, endothelial C3 and C5a expressions were required for endothelial activation by type 1 cytokines. Our data indicate that effective immunotherapy is a consequence of successful homing of T cells in response to local complement activation, which disrupts the tumor endothelial barrier.

A Tumor Mitochondria Vaccine Protects against Experimental Renal Cell Carcinoma.

Mitochondria provide energy for cells via oxidative phosphorylation. Reactive oxygen species, a byproduct of this mitochondrial respiration, can damage mitochondrial DNA (mtDNA), and somatic mtDNA mutations have been found in all colorectal, ovarian, breast, urinary bladder, kidney, lung, and pancreatic tumors studied. The resulting altered mitochondrial proteins or tumor-associated mitochondrial Ags (TAMAs) are potentially immunogenic, suggesting that they may be targetable Ags for cancer immunotherapy. In this article, we show that the RENCA tumor cell line harbors TAMAs that can drive an antitumor immune response. We generated a cellular tumor vaccine by pulsing dendritic cells with enriched mitochondrial proteins from RENCA cells. Our dendritic cell-based RENCA mitochondrial lysate vaccine elicited a cytotoxic T cell response in vivo and conferred durable protection against challenge with RENCA cells when used in a prophylactic or therapeutic setting. By sequencing mtDNA from RENCA cells, we identified two mutated molecules: COX1 and ND5. Peptide vaccines generated from mitochondrial-encoded COX1 but not from ND5 had therapeutic properties similar to RENCA mitochondrial protein preparation. Thus, TAMAs can elicit effective antitumor immune responses, potentially providing a new immunotherapeutic strategy to treat cancer.

Targeting tumor vasculature: expanding the potential of DNA cancer vaccines.

Targeting the tumor vasculature with anti-angiogenesis modalities is a bona fide validated approach that has complemented cancer treatment paradigms. Tumor vasculature antigens (TVA) can be immunologically targeted and offers multiple theoretical advantages that may enhance existing strategies against cancer. We focused on tumor endothelial marker 1 (TEM1/CD248) as a model TVA since it is broadly expressed on many different cancers. Our DNA-based vaccine approach demonstrated that CD248 can be effectively targeted immunologically; anti-tumor responses were generated in several mouse models; and CD8(+)/CD4(+) T cell responses were elicited against peptides derived from CD248 protein. Our work supports our contention that CD248 is a novel immunotherapeutic target for cancer treatment and highlights the efficient, safe and translatable use of DNA-based immunotherapy. We next briefly highlight ongoing investigations targeting CD248 with antibodies as a diagnostic imaging agent and as a therapeutic antibody in an early clinical trial. The optimal approach for generating effective DNA-based cancer vaccines for several tumor types may be a combinatorial approach that enhances immunogenicity such as combination with chemotherapy. Additional combination approaches are discussed and include those that alleviate the immunosuppressive tumor microenvironment induced by myeloid-derived suppressor cells and T regulatory cells. Targeting the tumor vasculature by CD248-based immunological modalities expands the armamentarium against cancer.

Tumor endothelial marker 1-specific DNA vaccination targets tumor vasculature.

Tumor endothelial marker 1 (TEM1; also known as endosialin or CD248) is a protein found on tumor vasculature and in tumor stroma. Here, we tested whether TEM1 has potential as a therapeutic target for cancer immunotherapy by immunizing immunocompetent mice with Tem1 cDNA fused to the minimal domain of the C fragment of tetanus toxoid (referred to herein as Tem1-TT vaccine). Tem1-TT vaccination elicited CD8+ and/or CD4+ T cell responses against immunodominant TEM1 protein sequences. Prophylactic immunization of animals with Tem1-TT prevented or delayed tumor formation in several murine tumor models. Therapeutic vaccination of tumor-bearing mice reduced tumor vascularity, increased infiltration of CD3+ T cells into the tumor, and controlled progression of established tumors. Tem1-TT vaccination also elicited CD8+ cytotoxic T cell responses against murine tumor-specific antigens. Effective Tem1-TT vaccination did not affect angiogenesis-dependent physiological processes, including wound healing and reproduction. Based on these data and the widespread expression of TEM1 on the vasculature of different tumor types, we conclude that targeting TEM1 has therapeutic potential in cancer immunotherapy.

Humanized NOD-SCID IL2rg­/­ mice as a preclinical model for cancer research and its potential use for individualized cancer therapies.

Humanized mouse models have been developed and utilized in cancer research for decades. Newly developed combined immunodeficient NOD-SCID-IL2rg­/­ mice are more permissive for human cells and tissue engraftment. In this review, we discuss the use of NOD-SCID-IL2rg(­/­) mice as a preclinical tool in cancer research and its potential use for individualized cancer therapies.

Changes in ovarian tumor cell number, tumor vasculature, and T cell function monitored in vivo using a novel xenograft model.

Despite an initial response to chemotherapy, most patients with ovarian cancer eventually progress and succumb to their disease. Understanding why effector T cells that are known to infiltrate the tumor do not eradicate the disease after cytoreduction is critically important to the development of novel therapeutic strategies to augment tumor immunity and improve patient outcomes. Such studies have been hampered by the lack of a suitable in vivo model. We report here a simple and reliable model system in which ovarian tumor cell aggregates implanted intraperitoneally into severely immunodeficient NSG mice establish tumor microenvironments within the omentum. The rapid establishment of tumor xenografts within this small anatomically well-defined site enables the recovery, characterization, and quantification of tumor and tumor-associated T cells. We validate here the ability of the omental tumor xenograft (OTX) model to quantify changes in tumor cell number in response to therapy, to quantify changes in the tumor vasculature, and to demonstrate and study the immunosuppressive effects of the tumor microenvironment. Using the OTX model, we show that the tumor-associated T cells originally present within the tumor tissues are anergic and that fully functional autologous T cells injected into tumor-bearing mice localize within the tumor xenograft. The transferred T cells remain functional for up to 3 days within the tumor microenvironment but become unresponsive to activation after 7 days. The OTX model provides for the first time the opportunity to study in vivo the cellular and molecular events contributing to the arrest in T cell function in human ovarian tumors.

Superior antitumor response induced by large stress protein chaperoned protein antigen compared with peptide antigen.

Our previous studies have demonstrated that the natural chaperone complexes of full-length tumor protein Ags (e.g., gp100) and large stress proteins (e.g., hsp110 and grp170) with exceptional Ag-holding capabilities augment potent tumor protective immunity. In this study, we assess the peptide-interacting property of these large chaperones and, for the first time, compare the immunogenicity of the recombinant chaperone vaccines targeting two forms of Ags (protein versus peptide). Both hsp110 and grp170 readily formed complexes with antigenic peptides under physiologic conditions, and the peptide association could be further stimulated by heat shock. The large chaperones displayed similar but distinct peptide-binding features compared with hsp70 and grp94/gp96. Immunization with hsp110- or grp170-tyrosinase-related protein 2 (TRP2(175-192)) peptide complexes effectively primed CD8(+) T cells reactive with TRP2-derived, MHC class I-restricted epitope. However, the tumor protective effect elicited by the TRP2(175-192) peptide vaccine was much weaker than that achieved by full-length TRP2 protein Ag chaperoned by grp170. Furthermore, immunization with combined chaperone vaccines directed against two melanoma protein Ags (i.e., gp100 and TRP2) significantly improved overall anti-tumor efficacy when compared with either of the single Ag vaccine. Lastly, treatment of tumor-bearing mice with these dual Ag-targeted chaperone complexes resulted in an immune activation involving epitope spreading, which was associated with a strong growth inhibition of the established tumors. Our results suggest that high m.w. chaperones are superior to conventional chaperones as a vaccine platform to deliver large protein Ags, and provide a rationale for translating this recombinant chaperoning-based vaccine to future clinical investigation.

Hsp110 and Grp170, members of the Hsp70 superfamily, bind to scavenger receptor-A and scavenger receptor expressed by endothelial cells-I.

Heat shock protein 110 (hsp110) and glucose-regulated protein (grp170) act as anti-cancer vaccines when complexed to tumor antigens by heat shock. It has been proposed that receptors on antigen-presenting cells contribute to HSP-mediated immune responses. Here, we show that hsp110 binds in a receptor-mediated manner to RAW264.7 macrophages, as does grp170. This hsp110/grp170 binding is inhibited by scavenger receptor ligands, suggesting a role for scavenger receptors as binding structures. We examined scavenger receptor class A (SR-A) and scavenger receptor expressed by endothelial cells-I (SREC-I). We show that hsp110/grp170 binds to both SR-A- and SREC-I-expressing CHO cells in a saturable manner and scavenger receptor ligands inhibit binding. Hsp110 also saturably binds mouse bone marrow-derived dendritic cells (bmDC) and is inhibited by scavenger receptor ligands. When an hsp110-rat neu (intracellular domain) heat shock complex vaccine is used to pulse mouse bmDC in vitro, an induction of IFN-gamma secretion is observed by CD8+ T lymphocytes isolated from vaccine-immunized mice. This immune response is inhibited by the application of scavenger receptor ligands to bmDC. Thus, SR-A and SREC-I appear to contribute to the binding of hsp110 and grp170 on APC. Scavenger receptors, in general, contribute to the cross-presentation of hsp110-chaperoned protein antigen.

Scavenger receptor-A negatively regulates antitumor immunity.

The scavenger receptor-A (SR-A), originally recognized by its ability to internalize modified lipoproteins, has largely been studied in relation to atherosclerosis as well as innate immunity against pathogen infection. SR-A was recently shown to be a receptor on antigen-presenting cell for heat shock protein (HSP) and was implicated in the cross-presentation of HSP-chaperoned antigens. Here, we show that SR-A is not required for antitumor immunity generated by HSP-based (e.g., grp170) vaccine approaches in vivo. The lack of SR-A significantly enhances HSP- or lipopolysaccharide-mediated vaccine activities against poorly immunogenic tumors, indicating that SR-A is able to attenuate immunostimulatory effects of adjuvants or "danger" molecules. The improved antitumor response in SR-A knockout mice is correlated with an increased antigen-specific T-cell response. Moreover, SR-A-deficient dendritic cells are more responsive to inflammatory stimuli and display a more effective antigen-presenting capability compared with wild-type cells. This is the first report illustrating that SR-A negatively regulates antigen-specific antitumor immunity, which has important clinical implications in vaccine design for cancer immunotherapy.

Immunoadjuvant chaperone, GRP170, induces 'danger signals' upon interaction with dendritic cells.

When chaperoning tumour antigens, glucose-regulated protein 170 (GRP170) is capable of inducing effective antitumour immune responses. In the present study, we determined whether such immunoadjuvant properties of GRP170 also involve the ability to induce 'danger signals' through interaction with APC. We prepared recombinant GRP170 in the baculovirus expression system with low endotoxin concentration at which LPS did not have any effect on dendritic cells (DC). We showed that GRP170 binds DC in a receptor-mediated fashion and induces DC to upregulate the expression of MHC class II, CD86 and CD40 molecules, and to secrete pro-inflammatory cytokines. GRP170 also induced expression of CD40 molecules in a B16F10 cell line, whereas LPS failed to do so. These findings show that GRP170 acts as a danger signal through its interaction with DC, regardless of its endotoxin component.

Chaperoning function of stress protein grp170, a member of the hsp70 superfamily, is responsible for its immunoadjuvant activity.

When used as vaccines, tumor-derived stress proteins can elicit antitumor immune responses. For members of the hsp70 superfamily, like grp170, this seems to be due to (a) the chaperoning of antigenic peptide by the stress protein and (b) the binding of the stress protein to receptor(s) on antigen-presenting cells (APC) and subsequent antigen presentation. This suggests that domains exist on the stress protein for each function. In this study, we determine the ability of grp170 and its structural domains to (a) bind to and present melanoma-associated antigen gp100 to the immune system and (b) to bind to receptors on APCs. A direct correlation between chaperone function, binding to APCs in a receptor-like manner, and antitumor immunity was observed. Two mutants that share no common sequence, yet are both effective in their antitumor activities, compete with one another for APC binding. Studies of other members of the hsp70 superfamily, hsp110 and hsp70, or their domain deletion mutants, further confirmed that APC binding segregates with chaperoning function and not sequence. Therefore, these studies suggest that molecular chaperoning is involved in stress protein interactions with APCs, antigen binding, and in eliciting antitumor immunity, thus bridging this ancient function of stress proteins in prokaryotes to their ability to elicit immunity in higher organisms.

Heat shock proteins HSP70 and GP96: structural insights.

Several heat shock proteins (HSPs) act as potent adjuvants for eliciting anti-tumor immunity. HSP-based tumor vaccine strategies have been highly successful in animal models and are undergoing testing in clinical trials. It is generally accepted that HSPs, functioning as chaperones for tumor antigens, elicit tumor-specific adaptive immune responses. HSPs also appear to induce innate immune responses in an antigen-independent fashion. Innate responses generated by HSPs may contribute to anti-tumor immunity. Immunologically active chaperones with anti-tumor activity are referred to as "immunochaperones". Here, we review the studies that address the role of structural domains or regions of the immunochaperones HSP70 and GP96 that may be involved in the induction of adaptive or innate immune responses.

Heat shock proteins and scavenger receptors: role in adaptive immune responses.

Tumor-derived heat shock proteins have shown promise as anti-cancer vaccines in clinical trials. Heat shock proteins (HSPs) can generate potent anti-tumor immunity and elicit antigen-specific CD8+ T cell responses in murine studies. Antigen presenting cells (APC), such as macrophages and dendritic cells (DCs), can elicit antigen-specific CD8+ T cell responses mediated by HSPs. CD91 was the first identified endocytic scavenger receptor for HSPs on APC that can facilitate the process of cross-presentation. Other scavenger receptors may also play a similar role in this process. The present review critically evaluates the identified HSP endocytic receptors on APCs that may generate adaptive immune responses. A better understanding of this interaction between HSPs and APCs may further unravel mechanisms of immunoadjuvant function of HSPs.

HSP110 induces "danger signals" upon interaction with antigen presenting cells and mouse mammary carcinoma.

HSP110 is a large molecular weight heat shock protein highly capable of chaperoning large proteins. When chaperoning tumour antigens, HSP110 is capable of eliciting effective anti-tumour immune responses. In the present study, we have determined whether such immunoadjuvant properties of HSP110 stem from its ability to induce "danger signals" through interaction with antigen presenting cells (APCs) and with tumour cells. In the previous studies, endotoxin contamination of HSP preparations was always a matter of concern and controversy. Therefore, we prepared recombinant HSP110 with low endotoxin concentration at which LPS did not have any effect on dendritic cells (DCs). We then evaluated the ability of the HSP110 to induce "danger signals" while interacting with APCs or mouse mammary carcinoma cell line (MMC), as evaluated by modulation of cell surface receptors and cytokines involved in innate and adaptive immune responses. We also performed competition studies in order to rule out contribution of endotoxin in HSP110 preparations while interacting with DCs and MMC. We showed that low endotoxin HSP110 induced DCs to up-regulate the expression of MHC class II, CD40 and CD86 molecules, and to secrete pro-inflammatory cytokines IL-6, IL-12 and TNF-alpha. Importantly, HSP110 induced MMC to secrete IL-12 and elevate secretion of IL-6 and expression of CD40 molecule. These findings demonstrate that HSP110 acts as a "danger signal" through its interaction with DCs and tumour cells, regardless of its endotoxin component. These immunoadjuvant properties of HSP110 suggest that pre-existing immunity in tumour-bearing individuals,may be due to the release of HSPs from tumours upon necrosis alerting the immune system against the tumours.

Immunotherapy of cancer using heat shock proteins.

Tumor derived heat shock protein (hsp)-peptide complexes (particularly hsp70 and grp94/gp96) have been demonstrated to serve as effective vaccines, producing anti-tumor immune responses in animals and in man. This approach utilizes the peptide binding properties of stress proteins which are responsible for their functions as molecular chaperones in numerous cellular processes. The present review briefly introduces the reader to the basic stress protein families, i.e. heat shock and glucose regulated proteins, their regulation, compartmentalization and family members. It then introduces the reader to aspects of hsps/grp function and interactions with the host's immune system. An overview of the conventional uses of hsp/grp vaccines as autologous vaccines derived from cancers is presented. We then discuss other stress protein related vaccination approaches. This includes the use of recombinant antigens, both proteins and peptides, naturally complexed to hsp/grps; hsp/grp DNA vaccines, hsp/grp fusion proteins and cell based hsp/grp vaccines. The advantages and disadvantages of each vaccination approach are discussed. Lastly, means of further enhancing the already potent activity of stress protein vaccines are presented, specifically the use of hyperthermia or CTLA-4 blockade as adjuvants.