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Nanoparticles awaken immune cells to fight cancer
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Science - By Robert F. ServiceJan. 5, 2017 , 5:00 PM
Tiny nanoparticles, far smaller than the width of a human hair, might help the body's own immune system fight tumors, a new study shows. In experiments with mice, the nanoparticle-based therapy not only wiped out the original targeted breast cancer tumors, but metastases in other parts of the body as well. Human clinical trials with the new therapy could begin within the next several months, researchers say.
The search for drugs that spur the immune system to fight tumors is one of the hottest fields in cancer research. Immune sentries, known as T cells, are normally on the prowl for suspicious looking targets, such as bacterial invaders and potential tumor cells. If they recognize one, they sound the alarm, inducing other immune cells to mount a larger response. However, the T cells' alarm can be muted by so-called immune checkpoints, other proteins on the surface of normal cells that tamp down the immune response to prevent harmful autoimmune reaction to normal tissue. Tumor cells often over express these checkpoint molecules, putting the brakes on the immune system's search and destroy work. To overcome that problem, pharmaceutical companies have developed a number of different antibody proteins that block these overexpressed checkpoint molecules and enable the immune system to target tumors. In cases where there are lots of T cells in the vicinity of a tumor, or where tumor cells have undergone large numbers of mutations, which creates additional targets for immune sentries, T cells will signal a full-fledged immune response to the cancer. Such cancer immunotherapy can add extra years to patients' lives.
However, existing cancer immunotherapy drugs work in only 20% to 30% of patients. In some cases, even when the checkpoint molecules are blocked that there are too few active T cells around to sound the immune alarm, says Jedd Wolchok, a cancer immunotherapy expert at the Memorial Sloan Kettering Cancer Center in New York City. In others, he says, tumors don't display enough of the T cell's targets, so-called tumor antigens, on their surface. But a seemingly unrelated puzzle offered the prospect of boosting immunotherapy's effectiveness. Oncologists have long known that in rare cases, after patients receive radiation therapy to shrink a tumor, the immune system will mount an aggressive response that wipes out not only the tumor, but metastases throughout the body that hadn't been treated with the radiation. Researchers now think that irradiation sometimes kills tumor cells in a manner that exposes new antigens to T cells, priming them to target other tumor cells that carry them as well, says Wenbin Lin, a chemist at the University of Chicago in Illinois, and one of the authors of the current study.
Lin wanted to see whether he could use nontoxic nanoparticles to sensitize the immune system in a similar way. Getting the nanoparticles themselves past the immune system isn't easy. If they're too big, cells in the blood called macrophages gobble them up. And blood proteins tend to coat the particles, facilitating their uptake. In recent years Lin's team devised a method to produce particles that are all between 20 and 40 nanometers in size (a nanometer is one-billionth of a meter), a range best able to elude macrophages. They also coated them with a polyethylene glycol shell, which helps them survive longer in blood circulation and enter target cells. Finally, on the inside they incorporated powerful light-absorbing, chlorine-based molecules that turn the nanoparticles into tumor killers. In previous studies, the team found that once injected into the bloodstream, the particles are able to circulate long enough to find their way in and around tumors. And because tumors typically have a leaky, ill-formed vasculature, the particles tend to leak out at the site of cancer tissue and be picked up and internalized inside tumor cells. Once the nanoparticles are absorbed, the researchers shine near infrared light on the tumors. That light is absorbed by the chlorine-based molecules, which then excite nearby oxygen molecules, creating a highly reactive form of oxygen, known as singlet oxygen, that rips apart nearby biomolecules and kills the tumor cell.
But that's only the start of it, Lin says. Singlet oxygen tends to rip apart tumor cells in a manner that exposes many new tumor antigens to immune cells called dendritic cells, which, like police executing a dragnet, grab the antigens and present them to T cells for closer inspection. By doing so they help the immune system mount a powerful antitumor response even in cases where there aren't that many T-cells nearby.
In August 2016, Lin and his colleagues reported in Nature Communications that when they injected a version of their nanoparticles into the bloodstream of mice with colon cancer along with a checkpoint antibody and blasted the tumors with light, the combination sparked the animals' immune systems to destroy both the targeted colon cancer tumors as well as untreated tumors elsewhere. However, those particles also ferried a standard chemotherapeutic toxin to help kill the cancer cells. In their current study the researchers wanted to see whether the approach would work with just the immune response. This time Lin and his colleagues worked with mice with breast cancer, another form of cancer that often doesn't respond to current immunotherapy drugs. Again, they injected the animals with their nanoparticles along with a checkpoint antibody. But this time their nanoparticles didn't contain any additional chemotherapeutic drug. They then blasted the tumors with infrared light, and waited for the results. And in almost every case, not only was the primary breast cancer tumor destroyed, but metastases in the lung were wiped out as well, they report in the Journal of the American Chemical Society. "We were surprised to find that without the cytotoxic agents, you can achieve the same effect," Lin says. "This is a well thought out approach, and the data is interesting," says Wolchok, who was not involved in the work. The approach deserves to be followed up with human trials, he adds. Lin says such trials are likely to start soon. The Chicago team has already formed a company, called Coordination Pharmaceuticals, which has raised seed funds to launch an early stage trial in humans, likely sometime in the second half of this year.
Core-shell nanoscale coordination polymers combine chemotherapy and photodynamic therapy to potentiate checkpoint blockade cancer immunotherapy
Advanced colorectal cancer is one of the deadliest cancers, with a 5-year survival rate of only 12% for patients with the metastatic disease. Checkpoint inhibitors, such as the antibodies inhibiting the PD-1/PD-L1 axis, are among the most promising immunotherapies for patients with advanced colon cancer, but their durable response rate remains low. We herein report the use of immunogenic nanoparticles to augment the antitumour efficacy of PD-L1 antibody-mediated cancer immunotherapy. Nanoscale coordination polymer (NCP) core-shell nanoparticles carry oxaliplatin in the core and the photosensitizer pyropheophorbide-lipid conjugate (pyrolipid) in the shell (NCP@pyrolipid) for effective chemotherapy and photodynamic therapy (PDT). Synergy between oxaliplatin and pyrolipid-induced PDT kills tumour cells and provokes an immune response, resulting in calreticulin exposure on the cell surface, antitumour vaccination and an abscopal effect. When combined with anti-PD-L1 therapy, NCP@pyrolipid mediates regression of both light-irradiated primary tumours and non-irradiated distant tumours by inducing a strong tumour-specific immune response.
Approximately 150,000 patients are diagnosed with colorectal cancer in the United States annually, with one-third dying from metastasis1. Although the 5-year survival rate for localized colorectal cancer is ∼89%, this number drops to only ∼12% for cancers that have metastasized to the liver, lungs or peritoneum2.
Stimulation of the host immune system has been shown to generate an antitumour immune response capable of controlling metastatic tumour growth3,4,5,6. Immune checkpoint blockade therapy, which targets regulatory pathways in T cells to enhance antitumour immune response, has witnessed significant clinical advances and provided a new strategy to combat cancer7. Among them, the PD-1/PD-L1 pathway inhibits immune activation by suppressing effector T-cell function8,9 and is upregulated in many tumours to cause apoptosis of tumour-specific cytotoxic T-lymphocytes and transmit an anti-apoptotic signal to tumour cells10,11. Antibody-mediated specific blockade of the PD-1/PD-L1 axis can generate potent antitumour activity in murine tumour models12,13. With the exception of metastatic melanoma, the durable responses generated by checkpoint blockade therapy are still low. Although blockade of PD-1 was shown not to be effective in metastatic colon cancer, a recent report by Le et al.14 demonstrated that PD-1 blockade was effective in a subset of colon cancer patients who were deficient in mismatch repair, reopening the door to immune modulation with interventions such as chemotherapy and radiotherapy to increase the durable response rate15. We hypothesize that combining PD-L1 blockade with multimodality nanoscale coordination polymer (NCP) nanoparticles can increase the response rate of checkpoint blockade cancer immunotherapy and perhaps broaden the use of immunotherapy in metastatic colon cancer.
As a new class of self-assembled hybrid nanomaterials composed of metal connecting points and organic bridging ligands16,17, NCPs have highly tunable compositions and structures, can combine multiple therapeutic agents or modalities18 and are intrinsically biodegradable. By combining non-toxic photosensitizers, light and oxygen to produce cytotoxic reactive oxygen species, in particular singlet oxygen (1O2), photodynamic therapy (PDT) kills cancer cells by apoptosis and necrosis, stimulates the host immune system and causes acute inflammation and leukocyte infiltration to the tumours, which increases the presentation of tumour-derived antigens to T cells19,20,21,22,23,24,25. Oxaliplatin was shown to induce immunogenic cell death (ICD) in murine colorectal cancer models26.
We herein report the design of NCP nanoparticles that carry oxaliplatin and the photosensitizer pyrolipid (NCP@pyrolipid), to significantly enhance antitumour immunity. NCP@pyrolipid combines two therapeutic modalities, chemotherapy and PDT, to elicit antitumour immunity27,28,29, as evidenced by early calreticulin (CRT) exposure on the cell surface, antitumour vaccination, tumour-specific T-cell response and an abscopal effect. The abscopal effect is usually described with ionizing radiation and refers to regression of tumour outside of the irradiated volume. Although the mechanism is unknown, it is thought to be immune modulated. More importantly, NCP@pyrolipid PDT treatment in combination with PD-L1 checkpoint blockade therapy not only led to the regression of the primary tumours, treated locally with light irradiation, but also resulted in the regression of the distant tumours in bilateral syngeneic mouse tumour models of CT26 and MC38 by generating systemic tumour-specific T-cell response with the infiltration of CD8+ T cells and CD4+ T cells in distant tumours.
As a significant percentage of patients with colorectal cancer die from the metastatic form of the disease37, it is critical to develop effective treatments that not only eradicate primary tumours but also control metastatic tumours. Our NCP-enabled regimen combines three treatment modalities-chemotherapy by oxaliplatin, PDT by pyrolipid and checkpoint blockade therapy with anti-PD-L1-to achieve superior anticancer efficacy in two syngeneic mouse models of colorectal cancer. NCP@pyrolipid can simultaneously kill cancer cells by inducing apoptosis and stimulate the immune system to activate both acute innate and prolonged adaptive immune responses via synergistic oxaliplatin chemotherapy and pyrolipid-based PDT. PDT of NCP@pyrolipid not only serves as an effective local therapy to eradicate/suppress primary tumour growth but also evokes systemic antitumour immunity, which further potentiates PD-L1 checkpoint blockade therapy.
Our understanding of cellular and molecular tumour immunology has evolved dramatically over the past two decades, which has enabled the identification of new and innovative ways to manipulate the immune response to cancer.38,39,40,41,42,43 Most immunotherapies target the immune system but not the cancer and, therefore, immunotherapies are believed to be a promising foundation to build treatment regimens for a variety of tumour types44,45,46,47. Clinical results suggest that immunotherapies have potential for durable and adaptable cancer control at different stages of the disease48. To maximize benefits, however, combination regimens with conventional cancer treatments that operate by distinct mechanisms will be necessary, to increase the durable response rate of cancer immunotherapies7.
NCP@pyrolipid nanoparticles self-assembled into core-shell structures with an asymmetric lipid bilayer coating that carried 27.6 wt% oxaliplatin in the core and released its cargo intracellularly. By passive targeting via the enhanced permeability and retention effect, NCP@pyrolipid achieved significantly higher cellular uptake of oxaliplatin and pyrolipid than other nanoparticle or free drug formulations. The efflux of oxaliplatin and pyrolipid was negligible, probably due to the partial incorporation of lipids into cell membranes during internalization that may have modified the membrane structure and prevented oxaliplatin or pyrolipid from effluxing out of the cells18. With optimal particle size, surface properties and stability, NCP@pyrolipid exhibited long blood circulation half-lives for both oxaliplatin and pyrolipid to leverage passive targeting, resulting in high tumour uptake of 10.4±0.7% ID g-1 24 h with low MPS clearance after i.p. injection.
Oxaliplatin is a Food and Drug Administration-approved chemotherapeutic drug for the treatment of colorectal cancer, known to induce cell death by triggering apoptosis and to stimulate pre-apoptotic CRT exposure, a distinct marker for ICD26,35. The exposure of CRT on the cell surface serves as an 'eat me' signal to dendritic cells and macrophages29. Mature dendritic cells migrate to the lymph node, where they prime naive T cells into effector T cells, which migrate to the tumour microenvironment34. NCP@pyrolipid leverages the immune system during chemotherapy by converting apoptotic death from 'silent' to immunogenic, thus acting as an 'anticancer vaccine.' The CRT exposure demonstrated by flow cytometry and CLSM, and successful prevention against tumour challenge by PDT of NCP@pyrolipid proved the effective ICD induced by the treatment.
As a local therapy, pyrolipid-enabled PDT33 also contributes to enhanced antitumour immunity by three mechanisms. First, PDT exerts systemic influence by promoting secretion of chemokines and cytokines, which stimulates the immune system to exert antitumour activity36. We observed significantly elevated pro-inflammatory cytokines TNF-α, INF-γ and IL-6 one day after PDT treatment, followed by a rapid drop in cytokine levels 2 days after PDT treatment (Fig. 3b-d), suggesting that the treatment evoked acute inflammation to prompt an innate immune response. Second, PDT has been found to induce ICD and thus activate the immune system36. Third, PDT of NCP@pyrolipid kills cancer cells by both apoptosis and necrosis (Supplementary Table 1). The innate immune effector cells engulf portions of the stressed and dying necrotic tumour cells and present tumour-derived antigenic peptides to T cells, thus stimulating a tumour-specific T-cell response36.
PDT of NCP@pyrolipid in combination with anti-PD-L1 treatment presents three regimens-oxaliplatin, PDT and checkpoint blockade therapy-to elicit synergistic effects in enhancing antitumour immunity for the effective treatment of metastatic colorectal cancer. PD-1 is a cell-surface co-inhibitory receptor expressed on T cells, B cells, monocytes and natural killer cells, and it has two known ligands, PD-L1 and PD-L2. PD-L1 is upregulated by tumour cells and by cells in the tumour microenvironment49. Multiple preclinical studies demonstrated that blockade of the interaction between PD-1 and PD-L1 using anti-PD-1 or anti-PD-L1 can restore T-cell activity against tumour cells, thereby preventing cancer metastasis and reducing tumour volume50,51. Infiltrating T cells and PD-L1 expression are essential for PD-L1 blockade therapy to be effective but are only found in immunogenic tumour microenvironment15,52,53. As a result, checkpoint blockade cancer therapy is only effective in patients whose tumours are immunogenic, which might explain the low rate of durable responses in clinical trials. We hypothesize that the response rate and efficacy of PD-L1 checkpoint blockade therapy can be improved when used in combination with therapies designed to create an immunogenic tumour microenvironment, eventually leading to durable clinical benefits. We believe that chemotherapy/PDT of NCP@pyrolipid provides an efficient way to induce immunogenicity in the tumour microenvironment and enhance antitumour immunity of anti-PD-L1 to empower checkpoint blockade cancer therapy.
We have elucidated the general principle that our NCP@pyrolipid can enhance the efficacy of PD-L1 checkpoint blockade therapy. The number of antigen-specific IFN-γ producing T cells and CD8+ T cells were significantly increased in tumour-bearing mice treated with NCP@pyrolipid with irradiation plus anti-PD-L1, as shown by ELISPOT assay, flow cytometry assay and inmmunofluorescence staining (Figs 7 and 8). Galon and coworkers54,55,56,57 have elegantly shown that the type, density and location of immune cells within human colorectal tumours are a better predictor of patient survival than the histopathological methods currently used to stage colorectal cancer. We thus intend to examine the changes in the tumour environment in our future work instead of the broader immune responses, although we also recognize that the immunological data in humans are considerably different to a subcutaneous colon cancer mouse model.
In summary, we have developed an effective NCP-enabled combination therapy for metastatic colorectal cancer that combined oxaliplatin chemotherapy, pyrolipid-based PDT and PD-L1 checkpoint blockade cancer therapy. NCP@pyrolipid carried high amounts of oxaliplatin and pyrolipid that showed prolonged blood circulation and favourable tumour accumulation after systemic administration. PDT of NCP@pyrolipid effectively inhibited tumour growth in subcutaneous CT26 and HT29 mouse models. More importantly, both oxaliplatin and PDT contributed to an immunogenic environment in the tumour, which significantly enhanced PD-L1 checkpoint blockade therapy by generating systemic antitumour immunity. As a result, PDT of NCP@pyrolipid in combination with anti-PD-L1 regressed the growth of not only primary tumours but also distant tumours in two bilateral syngeneic mouse models of colorectal cancer. We believe the combination of chemotherapy, PDT and checkpoint blockade therapy designed in the current study offer a new strategy for treating many metastatic cancers with primary tumours accessible by PDT.
Photodynamic Therapy Mediated by Nontoxic Core-Shell Nanoparticles Synergizes with Immune Checkpoint Blockade To Elicit Antitumor Immunity and Antimetastatic Effect on Breast

An effective, nontoxic, tumor-specific immunotherapy is the ultimate goal in the battle against cancer, especially the metastatic disease. Checkpoint blockade-based immunotherapies have been shown to be extraordinarily effective but benefit only the minority of patients whose tumors have been pre-infiltrated by T cells. Here, we show that Zn-pyrophosphate (ZnP) nanoparticles loaded with the photosensitizer pyrolipid (ZnP@pyro) can kill tumor cells upon irradiation with light directly by inducing apoptosis and/or necrosis and indirectly by disrupting tumor vasculature and increasing tumor immunogenicity. Furthermore, immunogenic ZnP@pyro photodynamic therapy (PDT) treatment sensitizes tumors to checkpoint inhibition mediated by a PD-L1 antibody, not only eradicating the primary 4T1 breast tumor but also significantly preventing metastasis to the lung. The abscopal effects on both 4T1 and TUBO bilateral syngeneic mouse models further demonstrate that ZnP@pyro PDT treatment combined with anti-PD-L1 results in the eradication of light-irradiated primary tumors and the complete inhibition of untreated distant tumors by generating a systemic tumor-specific cytotoxic T cell response. These findings indicate that nanoparticle-mediated PDT can potentiate the systemic efficacy of checkpoint blockade immunotherapies by activating the innate and adaptive immune systems in tumor microenvironment.


We have developed nontoxic and immunogenic ZnP@pyro nanoparticles for the effective treatment of metastatic breast cancer by combining PDT and checkpoint blockade immunotherapy. ZnP@pyro showed prolonged blood circulation and enhanced tumor accumulation after systemic administration, thereby effectively inhibiting tumor growth upon light irradiation. More importantly, ZnP@pyro-mediated PDT induced an immunogenic environment in tumors and sensitized tumors to PD-L1 checkpoint blockade therapy. As a result, ZnP@pyro PDT combined with anti-PD-L1 not only eradicated the primary tumors, but also prevented the lung metastasis and inhibited the pre-existing metastatic tumors by generating systemic antitumor immunity. Our results indicate that immunogenic therapies may provide immediate clinical benefit by expanding the small proportion of cancer patients who respond to current immune checkpoint treatments.
Breast cancer is the most common cancer for females in the United States and the second most common cause of cancer-related death in women.(1) In particular, metastatic triple-negative breast cancer (mTNBC) is associated with a poor prognosis and has no effective targeted therapy available, making this breast cancer subtype almost fatal.(2) The relative ineffectiveness of surgical interventions, radiation, and cytotoxic chemotherapies has driven interest in immunotherapy as a primary treatment modality.(3) Tumor immunotherapy operates on the premise that cancer cells can be eliminated by host cytotoxic CD8+ T cells,(4) although these cells themselves can be subjected to various suppressive mechanisms including inhibition by regulatory T (Treg) cells,(5) myeloid derived suppressor cells,(6) and induced expression of programmed death-1 (PD-1) and other inhibitory checkpoint receptors,(7) all limiting the antitumor functions of cytotoxic lymphocytes.
Targeting T cell inhibitory checkpoint signaling pathways overexpressed in tumors with antibodies has provided a promising strategy for tumor-specific immunotherapy.(8) The unusually high density of transmembrane protein PD-L1 expressed on tumors presents the PD-1/PD-L1 pathway as a valuable target:(9) two PD-1 targeted antibodies, nivolumab and pembrolizumab, and one PD-L1 targeted antibody, atezolizumab, have already been approved by the Food and Drug Administration for the treatments of advanced melanoma, non-small cell lung cancer, and bladder cancer, respectively.(10) However, only a small minority of cancer patients respond to checkpoint inhibition due to its reliance on high expression of PD-L1 on tumors and/or pre-existing tumor-infiltrating CD8+ T cells expressing PD-1.(7a, 11) This evidence indicates that strategies that can induce immunogenic tumor microenvironments to enhance T cell infiltration might sensitize tumors to checkpoint therapy and improve response rates.(4d, 12)
Photodynamic therapy (PDT) is a clinically used, minimally invasive therapeutic procedure that has also been shown to induce antitumor immunity.(13) In PDT, a photosensitizer (PS) accumulated in tumors is activated with a specific wavelength of light in the presence of oxygen to generate reactive oxygen species (ROS), predominantly the singlet oxygen (1O2), which kills tumor cells directly by inducing necrosis and/or apoptosis and indirectly by disrupting tumor vasculature and producing tumor-specific immunity.(14) The precise mechanisms involved in PDT-mediated induction of antitumor immunity are not yet fully understood. Potential contributing factors include alterations in the tumor microenvironment via stimulation of proinflammatory cytokines and direct effects of PDT on the tumor that increase immunogenicity.(15) We hypothesize that highly effective PDT can sensitize tumors to checkpoint blockade therapy by inducing acute inflammation and increasing tumor immunogenicity to broaden the use of checkpoint blockade immunotherapies in metastatic cancers.
Selective accumulation of PSs in tumors is critical for effective PDT by minimizing collateral damage to surrounding healthy tissues. However, typically PSs are hydrophobic and aggregate in aqueous media, which deleteriously affects their photophysical (decreased 1O2 formation), chemical (decreased solubility) and biological (insufficient tumor localization) properties, thereby diminishing the PDT efficacy.(16) Nanoparticles can increase the solubility of hydrophobic therapeutic or PDT agents and offer proper size and surface properties to prolong blood circulation, allowing for their selective accumulation in tumors via the enhanced permeability and retention (EPR) effect.(17) Tumor accumulation may be further improved by modifying the particle surface with cancer targeting ligands.(18) Indeed, a number of nanoparticles have been explored as promising delivery vehicles for molecule- or material-based PDT alone or combined with chemotherapeutic agents to cancers in order to enhance the phototreatment efficiency, and in some cases, encouraging preclinical and clinical data are emerging.(19)
Here we report the design of nontoxic core-shell nanoparticles (ZnP@pyro) with a coordination polymer of Zn and pyrophosphate (ZnP) in the core and the photosensitizer pyrolipid (a lipid conjugate of pyropheophorbide-a) in the shell for highly effective PDT. ZnP@pyro is optimally biocompatible as both Zn and pyrophosphate are endogenously found in blood plasma and pyrolipid is nontoxic without light activation.(20) The particles showed minimal uptake by the mononuclear phagocyte system (MPS), prolonged blood circulation, and preferential accumulation in the tumor after systemic injection, due to the EPR effect. The dual selectivity of tumor-targeted nanomedicine and the spatially controlled light irradiation minimizes damage to normal tissues to reduce systemic toxicity associated with classical PDT. This novel nanomedicine harnessed the power of PDT for direct cell killing and stimulation of systemic immune response for cancer treatment. We demonstrated that ZnP@pyro PDT treatment could sensitize tumors to checkpoint blockade therapy (Figure 1): the combination of ZnP@pyro PDT treatment with PD-L1 checkpoint blockade therapy not only eradicated the primary tumors, but also significantly prevented lung metastases in a 4T1 mTNBC murine model. In addition, the combination therapy produced an efficient abscopal effect on two bilateral syngeneic mouse models, 4T1 and TUBO, leading to the complete inhibition of the non-irradiated pre-existing distant tumors. These findings indicate that the proportion of cancers responding to checkpoint therapy can be substantially increased by combining checkpoint blockade with immunogenic conventional therapies such as PDT.

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