Jeremiah Oyer

Generation of Highly Cytotoxic Natural Killer Cells for Treatment of Acute Myelogenous Leukemia using a Feeder-Free, Particle-Based Approach

Natural killer (NK) cell immunotherapy as a cancer treatment shows promise, but expanding NK cells consistently from a small fraction (∼ 5%) of peripheral blood mononuclear cells (PBMCs) to therapeutic amounts remains challenging. Most current ex vivo expansion methods use co-culture with feeder cells (FC), but their use poses challenges for wide clinical application. We developed a particle-based NK cell expansion technology that uses plasma membrane particles (PM-particles) derived from K562-mbIL15-41BBL FCs. These PM-particles induce selective expansion of NK cells from unsorted PBMCs, with NK cells increasing 250-fold (median, 35; 10 donors; range, 94 to 1492) after 14 days of culture and up to 1265-fold (n = 14; range, 280 to 4426) typically after 17 days. The rate and efficiency of NK cell expansions with PM-particles and live FCs are comparable and far better than stimulation with soluble 41BBL, IL-15, and IL-2. Furthermore, NK cells expand selectively with PM-particles to 86% (median, 35; range, 71% to 99%) of total cells after 14 days. The extent of NK cell expansion and cell content was PM-particle concentration dependent. These NK cells were highly cytotoxic against several leukemic cell lines and also against patient acute myelogenous leukemia blasts. Phenotype analysis of these PM-particle-expanded NK cells was consistent with an activated cytotoxic phenotype. This novel NK cell expansion methodology has promising clinical therapeutic implications.

Cytokines in immunogenic cell death: Applications for cancer immunotherapy

&NA; Despite advances in treatments like chemotherapy and radiotherapy, metastatic cancer remains a leading cause of death for cancer patients. While many chemotherapeutic agents can efficiently eliminate cancer cells, long‐term protection against cancer is not achieved and many patients experience cancer recurrence. Mobilizing and stimulating the immune system against tumor cells is one of the most effective ways to protect against cancers that recur and/or metastasize. Activated tumor specific cytotoxic T lymphocytes (CTLs) can seek out and destroy metastatic tumor cells and reduce tumor lesions. Natural Killer (NK) cells are a front‐line defense against drug‐resistant tumors and can provide tumoricidal activity to enhance tumor immune surveillance. Cytokines like IFN‐&ggr; or TNF play a crucial role in creating an immunogenic microenvironment and therefore are key players in the fight against metastatic cancer. To this end, a group of anthracyclines or treatments like photodynamic therapy (PDT) exert their effects on cancer cells in a manner that activates the immune system. This process, known as immunogenic cell death (ICD), is characterized by the release of membrane‐bound and soluble factors that boost the function of immune cells. This review will explore different types of ICD inducers, some in clinical trials, to demonstrate that optimizing the cytokine response brought about by treatments with ICD‐inducing agents is central to promoting anti‐cancer immunity that provides long‐lasting protection against disease recurrence and metastasis. HighlightsShifting from inhibitory to activating cytokines is a challenge for immunotherapy.Cancer cells undergoing ICD act like “vaccines” to stimulate anti‐cancer immunity.Death induced by type I and II ICD inducers involve endoplasmic reticulum stress.Optimizing the cytokine response during ICD could yield new combination therapies.

The CT20 peptide causes detachment and death of metastatic breast cancer cells by promoting mitochondrial aggregation and cytoskeletal disruption

Metastasis accounts for most deaths from breast cancer, driving the need for new therapeutics that can impede disease progression. Rationally designed peptides that take advantage of cancer-specific differences in cellular physiology are an emerging technology that offer promise as a treatment for metastatic breast cancer. We developed CT20p, a hydrophobic peptide based on the C terminus of Bax that exhibits similarities with antimicrobial peptides, and previously reported that CT20p has unique cytotoxic actions independent of full-length Bax. In this study, we identified the intracellular actions of CT20p which precede cancer cell-specific detachment and death. Previously, we found that CT20p migrated in the heavy membrane fractions of cancer cell lysates. Here, using MDA-MB-231 breast cancer cells, we demonstrated that CT20p localizes to the mitochondria, leading to fusion-like aggregation and mitochondrial membrane hyperpolarization. As a result, the distribution and movement of mitochondria in CT20p-treated MDA-MB-231 cells was markedly impaired, particularly in cell protrusions. In contrast, CT20p did not associate with the mitochondria of normal breast epithelial MCF-10A cells, causing little change in the mitochondrial membrane potential, morphology or localization. In MDA-MB-231 cells, CT20p triggered cell detachment that was preceded by decreased levels of α5β1 integrins and reduced F-actin polymerization. Using folate-targeted nanoparticles to encapsulate and deliver CT20p to murine tumors, we achieved significant tumor regression within days of peptide treatment. These results suggest that CT20p has application in the treatment of metastatic disease as a cancer-specific therapeutic peptide that perturbs mitochondrial morphology and movement ultimately culminating in disruption of the actin cytoskeleton, cell detachment, and loss of cell viability.

Natural killer cells stimulated with PM21 particles expand and biodistribute in vivo: Clinical implications for cancer treatment

BACKGROUND AIMS Natural killer (NK) cell immunotherapy for treatment of cancer is promising, but requires methods that expand cytotoxic NK cells that persist in circulation and home to disease site. METHODS We developed a particle-based method that is simple, effective and specifically expands cytotoxic NK cells from peripheral blood mononuclear cells (PBMCs) both ex vivo and in vivo. This method uses particles prepared from plasma membranes of K562-mb21-41BBL cells, expressing 41BBL and membrane bound interleukin-21 (PM21 particles). RESULTS Ex vivo, PM21 particles caused specific NK-cell expansion from PBMCs from healthy donors (mean 825-fold, range 163-2216, n = 13 in 14 days) and acute myeloid leukemia patients. The PM21 particles also stimulated in vivo NK cell expansion in NSG mice. Ex vivo pre-activation of PBMCs with PM21 particles (PM21-PBMC) before intraperitoneal (i.p.) injection resulted in 66-fold higher amounts of hNK cells in peripheral blood (PB) of mice compared with unactivated PBMCs on day 12 after injection. In vivo administration of PM21 particles resulted in a dose-dependent increase of PB hNK cells in mice injected i.p. with 2.0 × 10(6) PM21-PBMCs (11% NK cells). Optimal dose of 800 µg/injection of PM21 particles (twice weekly) with low-dose interleukin 2 (1000 U/thrice weekly) resulted in 470 ± 40 hNK/µL and 95 ± 2% of total hCD45(+) cells by day 12 in PB. Furthermore, hNK cells were found in marrow, spleen, lung, liver and brain (day 16 after i.p. PM21/PBMC injection), and mice injected with PM21 particles had higher amounts. CONCLUSIONS The extent of NK cells observed in PB, their persistence and the biodistribution would be relevant for cancer treatment.

Conducting polymer nanoparticles for targeted cancer therapy

First and second generation photosensitizers used in photodynamic therapy (PDT) have shown promising results in clinical applications, aided by recent improvements in light absorption efficiency and quantum yield of singlet oxygen formation. However, these photosensitizers still have several drawbacks that prevent PDT from being an efficient therapy, including lack of selectivity to diseased tissue, observation of dark toxicity, and hydrophobicity of the sensitizer. Conducting polymers are promising candidates as next generation sensitizers for PDT due to their large extinction coefficients (>107 L mol−1 cm−1), ability to undergo intersystem crossing to the triplet state at high rates, and triplet energies that are close to that of oxygen. Targeting of conducting polymer poly[2-methoxy-5-(2-ethylhexyl-oxy)-p-phenylenevinylene] (MEH-PPV) nanoparticles to folate receptors (FR) was achieved by development of blended nanoparticles containing amphiphilic polymer polystyrene graft ethylene oxide functionalized with carboxylic acid (PS-PEG-COOH) with chemically active moieties that can be functionalized with folic acid. The resulting organic nanoparticles are buffer stable and exhibit excellent biocompatibility in the dark. The functionalized nanoparticles (FNPs) were studied in OVCAR3 (ovarian cancer cell line, FR+), MIA PaCa2 (pancreatic cell line, FR−), and A549 (lung cancer cell line, marginally FR+). Complete selectivity of the FNPs towards FR+ cell lines was found, and is attributed to the hydrophobicity and large negative zeta potential of the nanoparticles. Quantification of PDT results by MTS assays and flow cytometry show that PDT treatment was fully selective to the FR overexpressing cell line (OVCAR3). No cell mortality was observed for the other cell lines studied here within experimental error.

Anti-ovarian tumor response of donor peripheral blood mononuclear cells is due to infiltrating cytotoxic NK cells.

Treatment of ovarian cancer, a leading cause of gynecological malignancy, has good initial efficacy with surgery and platinum/taxane-based chemotherapy, but poor long-term survival in patients. Inferior long-term prognosis is attributed to intraperitoneal spreading, relapse and ineffective alternate therapies. Adoptive cell therapy is promising for tumor remission, although logistical concerns impede widespread implementation. In this study, healthy PBMCs were used to examine the immune response in a mouse model with human ovarian cancer, where natural killer (NK) cells were found to be the effector cells that elicited an anti-tumor response. Presence of tumor was found to stimulate NK cell expansion in mice treated intraperitoneally with PBMC+Interleukin-2 (IL-2), as compared to no expansion in non-tumor-bearing mice given the same treatment. PBMC+IL-2 treated mice exhibiting NK cell expansion had complete tumor remission. To validate NK cell mediated anti-tumor response, the intratumoral presence of NK cells and their cytotoxicity was confirmed by immunohistochemistry and granzyme activity of NK cells recovered from the tumor. Collectively, this study highlights the significance of NK cell-cytotoxic response to tumor, which may be attributed to interacting immune cell types in the PBMC population, as opposed to clinically used isolated NK cells showing lack of anti-tumor efficacy in ovarian cancer patients.

Difluoromethylornithine Combined with a Polyamine Transport Inhibitor Is Effective against Gemcitabine Resistant Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is highly chemo-resistant and has an extremely poor patient prognosis, with a survival rate at five years of <8%. There remains an urgent need for innovative treatments. Targeting polyamine biosynthesis through inhibition of ornithine decarboxylase with difluoromethylornithine (DFMO) has had mixed clinical success due to tumor escape via an undefined transport system, which imports exogenous polyamines and sustains intracellular polyamine pools. Here, we tested DFMO in combination with a polyamine transport inhibitor (PTI), Trimer44NMe, against Gemcitabine-resistant PDAC cells. DFMO alone and with Trimer44NMe significantly reduced PDAC cell viability by inducing apoptosis or diminishing proliferation. DFMO alone and with Trimer44NMe also inhibited in vivo orthotopic PDAC growth and resulted in decreased c-Myc expression, a readout of polyamine pathway dysfunction. Moreover, dual inhibition significantly prolonged survival of tumor-bearing mice. Collectively, these studies demonstrate that targeting polyamine biosynthesis and import pathways in PDAC can lead to increased survival in pancreatic cancer.

Comparison of the cytotoxic response against ovarian cancer by immune effector cells isolated and expanded from normal donors and ovarian cancer patients

BACKGROUND/AIMS The aim of this study was to compare the cytotoxic response against ovarian cancer (OC) cells elicited by different immune effector cells in combination with the cytokines interleukin (IL)-2 and interferon (IFN) α-2b. METHODS OC cells were co-cultured with peripheral blood mononuclear cells (PBMC) from normal donors or OC patients and IL-2 or IFN α-2b alone or in combination, in order to determine the cytotoxicity. T cells were isolated from healthy donors to determine T cell cytotoxic activity. PBMC from healthy donors and OC patients were expanded in an IL-2/IL-7/IL-12 cocktail with and without anti-CD3 antibody, and the cytotoxic activity measured. Flow cytometry was performed on primary, selected and expanded cells to determine T, B, and natural killer- (NK) cell percentages. RESULTS Healthy donor PBMC elicited a significant cytotoxic response (59%) compared with OC patient PBMC (7%). T cells enriched from normal donors elicited a significant cytotoxic response (18%) compared with controls lacking effector cells (1.4%); however, the cytotoxicity observed was significantly less compared with unselected PBMC. Expanded effector cells consisted primarily of T cells (98%) and the fold-expansion was significantly higher in the presence of anti-CD3 (19- versus 132-fold). No significant difference in the expansion (either fold-expansion or cell type) was observed between OC patients and healthy donors. Expanded cells from both healthy donors and OC patients elicited a significant cytotoxic response in the presence of IL-2 (19% and 22%) compared with controls. CONCLUSIONS PBMC from OC patients do not elicit a significant cytotoxic response; however, ex vivo-expanded cells from OC patients are capable of cytotoxic killing similar to unexpanded T cells isolated from normal donors. These data provide the groundwork for further development of cellular therapy against OC.

PD-L1 blockade enhances anti-tumor efficacy of NK cells

ABSTRACT Anti-PD-1/anti-PD-L1 therapies have shown success in cancer treatment but responses are limited to ~ 15% of patients with lymphocyte infiltrated, PD-L1 positive tumors. Hence, strategies that increase PD-L1 expression and tumor infiltration should make more patients eligible for PD-1/PD-L1 blockade therapy, thus improving overall outcomes. PD-L1 expression on tumors is induced by IFNγ, a cytokine secreted by NK cells. Therefore, we tested if PM21-particle expanded NK cells (PM21-NK cells) induced expression of PD-L1 on tumors and if anti-PD-L1 treatment enhanced NK cell anti-tumor efficacy in an ovarian cancer model. Studies here showed that PM21-NK cells secrete high amounts of IFNγ and that adoptively transferred PM21-NK cells induce PD-L1 expression on SKOV-3 cells in vivo. The induction of PD-L1 expression on SKOV-3 cells coincided with the presence of regulatory T cells (Tregs) in the abdominal cavity and within tumors. In in vitro experiments, anti-PD-L1 treatment had no direct effect on cytotoxicity or cytokine secretion by predominantly PD-1 negative PM21-NK cells in response to PD-L1+ targets. However, significant improvement of NK cell anti-tumor efficacy was observed in vivo when combined with anti-PD-L1. PD-L1 blockade also resulted in increased in vivo NK cell persistence and retention of their cytotoxic phenotype. These results support the use of anti-PD-L1 in combination with NK cell therapy regardless of initial tumor PD-L1 status and indicate that NK cell therapy would likely augment the applicability of anti-PD-L1 treatment.

Abstract A47: Use of a cytotoxic peptide that induces immunogenic cell death to engage innate immunity in the treatment of metastatic breast cancer

The median survival rate for women with metastatic breast cancer is 1-2 years, and the 5-year survival rate is only 15 percent. Early detection of metastatic disease is often ineffective, since once the cancer recurs at a distant site, it may be refractory to conventional treatments. A promising strategy for targeting metastatic breast cancer is engaging the immune system to destroy disseminated breast cancer cells. Immunotherapeutic approaches are effective treatments for many types of cancer, such as melanoma. However, breast cancer is a challenge for immunotherapy because of its inherent genetic heterogeneity and decreased immunogenicity. Moreover, immunotherapy can cause adverse side effects such as colitis. There is an unmet need to develop new, targeted therapies for breast cancer that stimulate the immune system to fight metastatic disease. One potential approach is to generate dying breast cancer cells that operate like a vaccine to induce a tumor-specific immune response, which can eradicate residual cancer cells. This is termed immunogenic cell death (ICD) and is characterized by a unique molecular signature, involving the release of molecules that attract and stimulate phagocytes like dendritic cells (DCs) that in turn activate tumor-specific cytotoxic T cells. While not well-understood, ICD may also sensitize tumor cells to killing by natural killer (NK) cells. With the discovery of new ICD-inducing agents, interest in this approach is increasing. However, the current doses of drugs, such as anthracyclines, used to induce ICD may be too high to translate into clinically relevant regimens. To address these problems, our group developed a novel cytotoxic peptide, CT20p, and a nanotechnology-based platform to deliver and concentrate CT20p in breast tumors. We found that treatment of mice with nanomolar amounts of CT20p, encapsulated in nanoparticles formed with a novel hyperbranched polyester polymer (HBPE-NPs), resulted in regression of breast cancer tumors, and that dying breast cancer cells expressed markers characteristic of ICD, such as the pre-mortem exposure of calreticulin (Crt). The intracellular target of CT20p is a protein called chaperonin-containing T-complex (CCT) which is essential for the folding of actin and tubulin and other critical proteins into their native forms. We found CCT to be overexpressed in breast cancer cells susceptible to the peptide. Inhibition of CCT activity by CT20p, indicated by decreased F-actin, disrupted the cytoskeleton, causing loss of cell motility and adhesion that led to breast cancer cell death. Disruption of CCT could cause endoplasmic reticulum (ER) stress through the accumulation of misfolded proteins. ER stress in turn initiates intracellular pathways that can generate the danger signals associate with ICD. To this end, we observed that breast cancer cells treated with CT20p displayed alterations in PERK (protein kinase RNA-like ER kinase), one of the key mediators of the unfolded protein response (UPR). As a result, CT20p-treated breast cancer cells were more readily phagocytosed. This also suggests that peptide treatment could potentially have an impact on NK cell cytotoxicity by inducing the expression of activating ligands on cancer cells. In our experiments normal breast epithelial cells, macrophages or NK cells were unaffected by CT20p treatment due reduced levels and activity of CCT. These studies indicate that CT20p, by targeting CCT, can be used to induce ICD and stimulate essential innate immune functions like NK cell cytotoxic activity and leukocyte phagocytosis that are necessary for promoting a robust anti-cancer adaptive immune response in order to mitigate the development of lethal metastatic disease. Citation Format: Arati Limaye, Rania Bassiouni, Jeremiah Oyer, Robert W. Igarashi, Orielyz Flores, J. Manuel Perez, Alijca Copik, Annette R. Khaled. Use of a cytotoxic peptide that induces immunogenic cell death to engage innate immunity in the treatment of metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A47.