Unleashing Type-2 Dendritic Cells to Drive Protective Antitumor CD4(+) T Cell Immunity
Differentiation of proinflammatory CD4(+) conventional T cells (Tconv) is critical for productive antitumor responses yet their elicitation remains poorly understood. We comprehensively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4(+) Tconv, but then fail to support antitumor CD4(+) Tconv differentiation. Regulatory T cell (Treg) depletion enhanced their capacity to elicit strong CD4(+) Tconv responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice, their abundance relative to Treg predicts protective ICOS(+) PD-1(lo) CD4(+) Tconv phenotypes and survival. Further, in melanoma patients with low Treg abundance, intratumoral cDC2 density alone correlates with abundant CD4(+) Tconv and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway that restrains cDC2 and whose reversal enhances CD4(+) Tconv abundance and controls tumor growth.
Systemic clinical tumor regressions and potentiation of PD1 blockade with in situ vaccination
Indolent non-Hodgkin's lymphomas (iNHLs) are incurable with standard therapy and are poorly responsive to checkpoint blockade. Although lymphoma cells are efficiently killed by primed T cells, in vivo priming of anti-lymphoma T cells has been elusive. Here, we demonstrate that lymphoma cells can directly prime T cells, but in vivo immunity still requires cross-presentation. To address this, we developed an in situ vaccine (ISV), combining Flt3L, radiotherapy, and a TLR3 agonist, which recruited, antigen-loaded and activated intratumoral, cross-presenting dendritic cells (DCs). ISV induced anti-tumor CD8(+) T cell responses and systemic (abscopal) cancer remission in patients with advanced stage iNHL in an ongoing trial ( NCT01976585 ). Non-responding patients developed a population of PD1(+)CD8(+) T cells after ISV, and murine tumors became newly responsive to PD1 blockade, prompting a follow-up trial of the combined therapy. Our data substantiate that recruiting and activating intratumoral, cross-priming DCs is achievable and critical to anti-tumor T cell responses and PD1-blockade efficacy.
Targeted antibody and cytokine cancer immunotherapies through collagen affinity
Cancer immunotherapy with immune checkpoint inhibitors (CPIs) and interleukin-2 (IL-2) has demonstrated clinical efficacy but is frequently accompanied with severe adverse events caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-cytotoxic T lymphocyte antigen 4 antibody (alphaCTLA4) + anti-programmed death ligand 1 antibody (alphaPD-L1) and the cytokine IL-2 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokine) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain, harnessing the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously administered CBD protein accumulated mainly in tumors. CBD conjugation or fusion decreases the systemic toxicity of both alphaCTLA4 + alphaPD-L1 combination therapy and IL-2, for example, eliminating hepatotoxicity with the CPI molecules and ameliorating pulmonary edema with IL-2. Both CBD-CPI and CBD-IL-2 suppressed tumor growth compared to their unmodified forms in multiple murine cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8(+) T cells. In an orthotopic breast cancer model, combination treatment with CPI and IL-2 eradicated tumors in 9 of 13 animals with the CBD-modified drugs, whereas it did so in only 1 of 13 animals with the unmodified drugs. Thus, the A3 domain of VWF can be used to improve safety and efficacy of systemically administered tumor drugs with high translational promise.
CD91 on dendritic cells governs immunosurveillance of nascent, emerging tumors
The immune system detects aberrant, premalignant cells and eliminates them before the development of cancer. Immune cells, including T cells, have been shown to be critical components in eradicating these aberrant cells, and when absent in the host, incidence of cancer increases. Here, we show that CD91, a receptor expressed on antigen-presenting cells, is required for priming immune responses to nascent, emerging tumors. In the absence of CD91, effector immune responses are subdued, and tumor incidence and progression are amplified. We also show that, consequently, tumors that arise in the absence of CD91 express neo-epitopes with indices that are indicative of greater immunogenicity. Polymorphisms in human CD91 that are expected to affect ligand binding are shown to influence antitumor immune responses in cancer patients. This study presents a molecular mechanism for priming immune responses to nascent, emerging tumors that becomes a predictor of cancer susceptibility and progression.
A designer self-assembled supramolecule amplifies macrophage immune responses against aggressive cancer
Effectively activating macrophages that can 'eat' cancer cells is challenging. In particular, cancer cells secrete macrophage colony stimulating factor (MCSF), which polarizes tumour-associated macrophages from an antitumour M1 phenotype to a pro-tumourigenic M2 phenotype. Also, cancer cells can express CD47, an 'eat me not' signal that ligates with the signal regulatory protein alpha (SIRPalpha) receptor on macrophages to prevent phagocytosis. Here, we show that a supramolecular assembly consisting of amphiphiles inhibiting the colony stimulating factor 1 receptor (CSF-1R) and displaying SIRPalpha-blocking antibodies with a drug-to-antibody ratio of 17,000 can disable both mechanisms. The supramolecule homes onto SIRPalpha on macrophages, blocking the CD47-SIRPalpha signalling axis while sustainedly inhibiting CSF-1R. The supramolecule enhances the M2-to-M1 repolarization within the tumour microenvironment, and significantly improves antitumour and antimetastatic efficacies in two aggressive animal models of melanoma and breast cancer, with respect to clinically available small-molecule and biologic inhibitors of CSF-1R signalling. Simultaneously blocking the CD47-SIRPalpha and MCSF-CSF-1R signalling axes may constitute a promising immunotherapy.
Unlocking the therapeutic potential of primary tumor-draining lymph nodes
Lymph nodes draining the primary tumor are essential for the initiation of an effective anti-tumor T-cell immune response. However, cancer-derived immune suppressive factors render the tumor-draining lymph nodes (TDLN) immune compromised, enabling tumors to invade and metastasize. Unraveling the different mechanisms underlying this immune escape will inform therapeutic intervention strategies to halt tumor spread in early clinical stages. Here, we review our findings from translational studies in melanoma, breast, and cervical cancer and discuss clinical opportunities for local immune modulation of TDLN in each of these indications.
Suppression of Exosomal PD-L1 Induces Systemic Anti-tumor Immunity and Memory
PD-L1 on the surface of tumor cells binds its receptor PD-1 on effector T cells, thereby suppressing their activity. Antibody blockade of PD-L1 can activate an anti-tumor immune response leading to durable remissions in a subset of cancer patients. Here, we describe an alternative mechanism of PD-L1 activity involving its secretion in tumor-derived exosomes. Removal of exosomal PD-L1 inhibits tumor growth, even in models resistant to anti-PD-L1 antibodies. Exosomal PD-L1 from the tumor suppresses T cell activation in the draining lymph node. Systemically introduced exosomal PD-L1 rescues growth of tumors unable to secrete their own. Exposure to exosomal PD-L1-deficient tumor cells suppresses growth of wild-type tumor cells injected at a distant site, simultaneously or months later. Anti-PD-L1 antibodies work additively, not redundantly, with exosomal PD-L1 blockade to suppress tumor growth. Together, these findings show that exosomal PD-L1 represents an unexplored therapeutic target, which could overcome resistance to current antibody approaches.
TLR3 Activation of Intratumoral CD103(+) Dendritic Cells Modifies the Tumor Infiltrate Conferring Anti-tumor Immunity
An important challenge in cancer immunotherapy is to expand the number of patients that benefit from immune checkpoint inhibitors (CI), a fact that has been related to the pre-existence of an efficient anti-tumor immune response. Different strategies are being proposed to promote tumor immunity and to be used in combined therapies with CI. Recently, we reported that intratumoral administration of naked poly A:U, a dsRNA mimetic empirically used in early clinical trials with some success, delays tumor growth and prolongs mice survival in several murine cancer models. Here, we show that CD103(+) cDC1 and, to a much lesser extent CD11b(+) cDC2, are the only populations expressing TLR3 at the tumor site, and consequently could be potential targets of poly A:U. Upon poly A:U administration these cells become activated and elicit profound changes in the composition of the tumor immune infiltrate, switching the immune suppressive tumor environment to anti-tumor immunity. The sole administration of naked poly A:U promotes striking changes within the lymphoid compartment, with all the anti-tumoral parameters being enhanced: a higher frequency of CD8(+) Granzyme B(+) T cells, (lower Treg/CD8(+) ratio) and an important expansion of tumor-antigen specific CD8(+) T cells. Also, PD1/PDL1 showed an increased expression indicating that neutralization of this axis could be exploited in combination with poly A:U. Our results shed new light to promote further assays in this dsRNA mimetic to the clinical field.
Nanobody-based CAR T cells that target the tumor microenvironment inhibit the growth of solid tumors in immunocompetent mice
Chimeric antigen receptor (CAR) T cell therapy has been successful in clinical trials against hematological cancers, but has experienced challenges in the treatment of solid tumors. One of the main difficulties lies in a paucity of tumor-specific targets that can serve as CAR recognition domains. We therefore focused on developing VHH-based, single-domain antibody (nanobody) CAR T cells that target aspects of the tumor microenvironment conserved across multiple cancer types. Many solid tumors evade immune recognition through expression of checkpoint molecules, such as PD-L1, that down-regulate the immune response. We therefore targeted CAR T cells to the tumor microenvironment via the checkpoint inhibitor PD-L1 and observed a reduction in tumor growth, resulting in improved survival. CAR T cells that target the tumor stroma and vasculature through the EIIIB(+) fibronectin splice variant, which is expressed by multiple tumor types and on neovasculature, are likewise effective in delaying tumor growth. VHH-based CAR T cells can thus function as antitumor agents for multiple targets in syngeneic, immunocompetent animal models. Our results demonstrate the flexibility of VHH-based CAR T cells and the potential of CAR T cells to target the tumor microenvironment and treat solid tumors.
Upstream Position of Proline Defines Peptide-HLA Class I Repertoire Formation and CD8(+) T Cell Responses
Cytotoxic CD8(+) T lymphocytes (CTLs) recognize peptides displayed by HLA class I molecules on cell surfaces, monitoring pathological conditions such as cancer. Difficulty in predicting HLA class I ligands is attributed to the complexity of the Ag processing pathway across the cytosol and the endoplasmic reticulum. By means of HLA ligandome analysis using mass spectrometry, we collected natural HLA class I ligands on a large scale and analyzed the source-protein sequences flanking the ligands. This comprehensive analysis revealed that the frequency of proline at amino acid positions 1-3 upstream of the ligands was selectively decreased. The depleted proline signature was the strongest among all the upstream and downstream profiles. Experiments using live cells demonstrated that the presence of proline at upstream positions 1-3 attenuated CTL responses against a model epitope. Other experiments, in which N-terminal-flanking Ag precursors were confined in the endoplasmic reticulum, demonstrated an inability to remove upstream prolines regardless of their positions, suggesting a need for synergistic action across cellular compartments for making the proline signature. Our results highlight, to our knowledge, a unique role and position of proline for inhibiting downstream epitope presentation, which provides a rule for defining natural peptide-HLA class I repertoire formation and CTL responses.