PM21-particle stimulation augmented with cytokines enhances NK cell expansion and confers memory-like characteristics with enhanced survival.

NK cell therapeutics have gained significant attention as a potential cancer treatment. Towards therapeutic use, NK cells need to be activated and expanded to attain high potency and large quantities for an effective dosage. This is typically done by ex vivo stimulation with cytokines to enhance functionality or expansion for 10-14 days to increase both their activity and quantity. Attaining a robust methodology to produce large doses of potent NK cells for an off-the-shelf product is highly desirable. Notably, past reports have shown that stimulating NK cells with IL-12, IL-15, and IL-18 endows them with memory-like properties, better anti-tumor activity, and persistence. While this approach produces NK cells with clinically favorable characteristics supported by encouraging early results for the treatment of hematological malignancies, its limited scalability, variability in initial doses, and the necessity for patient-specific production hinder its broader application. In this study, stimulation of NK cells with PM21-particles derived from K562-41BBL-mbIL21 cells was combined with memory-like induction using cytokines IL-12, IL-15, and IL-18 to produce NK cells with enhanced anti-tumor function. The use of cytokines combined with PM21-particles (cytokine and particle, CAP) significantly enhanced NK cell expansion, achieving a remarkable 8,200-fold in 14 days. Mechanistically, this significant improvement over expansion with PM21-particles alone was due to the upregulation of receptors for key stimulating ligands (4-1BBL and IL-2), resulting in a synergy that drives substantial NK cell growth, showcasing the potential for more effective therapeutic applications. The therapeutic potential of CAP-NK cells was demonstrated by the enhanced metabolic fitness, persistence, and anti-tumor function both in vitro and in vivo. Finally, CAP-NK cells were amenable to current technologies used in developing therapeutic NK cell products, including CRISPR/Cas9-based techniques to generate a triple-gene knockout or a gene knock-in. Taken together, these data demonstrate that the addition of cytokines enhanced the already effective method of ex vivo generation of therapeutic NK cells with PM21-particles, yielding a superior NK cell product for manufacturing efficiency and potential therapeutic applications.

Adoptive T cell therapy for ovarian cancer.

Although ovarian cancer patients typically respond to standard of care therapies, including chemotherapy and DNA repair inhibitors, the majority of tumors recur highlighting the need for alternative therapies. Ovarian cancer is an immunogenic cancer in which the accumulation of tumor infiltrating lymphocytes (TILs), particularly T cells, is associated with better patient outcome. Thus, harnessing the immune system through passive administration of T cells, a process called adoptive cell therapy (ACT), is a promising therapeutic option for the treatment of ovarian cancer. There are multiple routes by which tumor-specific T cell products can be generated. Dendritic cell cancer vaccines can be administered to the patients to induce or bolster T cell responses against tumor antigens or be utilized ex vivo to prime T cells against tumor antigens; these T cells can then be prepared for infusion. ACT protocols can also utilize naturally-occurring tumor-reactive T cells isolated from a patient tumor, known as TILs, as these cells often are heterogeneous in composition and antigen specificity with patient-specific cancer recognition. Alternatively, T cells may be sourced from the peripheral blood, including those that are genetically modified to express a tumor antigen-specific T cell receptor (TCR) or chimeric antigen receptor (CAR) to redirect their specificity and promote their activity against tumor cells expressing the target tumor antigen. Here, we review current ACT strategies for ovarian cancer and provide insights into advancing ACT therapy strategies for the treatment of ovarian cancer.

CD137+ tumor infiltrating lymphocytes predicts ovarian cancer survival.

OBJECTIVE: Ovarian cancer (OC) is the leading cause of death from gynecologic malignancy in the United States, and biomarkers of patient outcomes are limited. Data using immunohistochemical (IHC) analysis are mixed regarding whether and which tumor infiltrating lymphocytes (TILs) impact survival, and IHC does not adequately quantify rare cell populations, including CD137+ (4-1BB) tumor-reactive TILs. Our study investigates if a higher percentage of CD3+ CD137+ TILs is associated with improved overall survival (OS) in OC. METHODS: Flow cytometry was performed on viably banked OC digests. Chart review and statistical analysis were performed. Forty-seven patients were included, 40 of whom were diagnosed with high-grade serous ovarian carcinoma (HGSOC), papillary serous carcinoma, or undifferentiated histology. RESULTS: A high percentage of CD3+ CD137+ TILs correlated with improved OS (n = 40, r = 0.48, P = 0.0016). Subjects were divided into CD3+ CD137+ TIL high and low groups by the median. Subjects with high CD3+CD137+ TIL frequencies (>9.6%) had longer OS (Wilcoxon rank-sum test; P = 0.0032) and improved OS (logrank test; P = 0.007). Differences in CD3+ or CD3+ CD8+ TILs did not impact survival. CD3+ CD137+ TILs were predictive of OS regardless of germline mutation or debulking status. Analysis of subgroups including late stage HGSOC and late stage HGSOC with primary optimal cytoreduction indicated CD3+ CD137+ TILs correlated with improved OS after adjusting for age and PARP inhibitor use (P = 0.034 and P = 0.016, respectively). CONCLUSIONS: Prevalence of CD3+ CD137+ TILs in digested OC specimens is associated with improved OS, while general TIL markers are not. CD137 has the potential to be a novel biomarker for survival in OC.

A B7-H4-Targeting Antibody-Drug Conjugate Shows Antitumor Activity in PARPi and Platinum-Resistant Cancers with B7-H4 Expression.

PURPOSE: Platinum and PARP inhibitors (PARPi) demonstrate activity in breast and ovarian cancers, but drug resistance ultimately emerges. Here, we examine B7-H4 expression in primary and recurrent high-grade serous ovarian carcinoma (HGSOC) and the activity of a B7-H4-directed antibody-drug conjugate (B7-H4-ADC), using a pyrrolobenzodiazepine-dimer payload, in PARPi- and platinum-resistant HGSOC patient-derived xenograft (PDX) models. EXPERIMENTAL DESIGN: B7-H4 expression was quantified by flow cytometry and IHC. B7-H4-ADC efficacy was tested against multiple cell lines in vitro and PDX in vivo. The effect of B7-H4-ADC on cell cycle, DNA damage, and apoptosis was measured using flow cytometry. RESULTS: B7-H4 is overexpressed in 92% of HGSOC tumors at diagnosis (n = 12), persisted in recurrent matched samples after platinum treatment, and was expressed at similar levels across metastatic sites after acquired multi-drug resistance (n = 4). Treatment with B7-H4-ADC resulted in target-specific growth inhibition of multiple ovarian and breast cancer cell lines. In platinum- or PARPi-resistant ovarian cancer cells, B7-H4-ADC significantly decreased viability and colony formation while increasing cell-cycle arrest and DNA damage, ultimately leading to apoptosis. Single-dose B7-H4-ADC led to tumor regression in 65.5% of breast and ovarian PDX models (n = 29), with reduced activity in B7-H4 low or negative models. In PARPi and platinum-resistant HGSOC PDX models, scheduled B7-H4-ADC dosing led to sustained tumor regression and increased survival. CONCLUSIONS: These data support B7-H4 as an attractive ADC target for treatment of drug-resistant HGSOC and provide evidence for activity of an ADC with a DNA-damaging payload in this population. See related commentary by Veneziani et al., p. 1434.

Dual blockade of BRD4 and ATR/WEE1 pathways exploits ARID1A loss in clear cell ovarian cancer.

ARID1A, an epigenetic tumor suppressor, is the most common gene mutation in clear-cell ovarian cancers (CCOCs). CCOCs are often resistant to standard chemotherapy and lack effective therapies. We hypothesized that ARID1A loss would increase CCOC cell dependency on chromatin remodeling and DNA repair pathways for survival. We demonstrate that combining BRD4 inhibitor (BRD4i) with DNA damage response inhibitors (ATR or WEE1 inhibitors; e.g. BRD4i-ATRi) was synergistic at low doses leading to decreased survival, and colony formation in CCOC in an ARID1A dependent manner. BRD4i-ATRi caused significant tumor regression and increased overall survival in ARID1AMUT but not ARID1AWT patient-derived xenografts. Combination BRD4i-ATRi significantly increased γH2AX, and decreased RAD51 foci and BRCA1 expression, suggesting decreased ability to repair DNA double-strand-breaks (DSBs) by homologous-recombination in ARID1AMUT cells, and these effects were greater than monotherapies. These studies demonstrate BRD4i-ATRi is an effective treatment strategy that capitalizes on synthetic lethality with ARID1A loss in CCOC.

Leveraging immune resistance archetypes in solid cancer to inform next-generation anticancer therapies.

Anticancer immunotherapies, such as immune checkpoint inhibitors, bispecific antibodies, and chimeric antigen receptor T cells, have improved outcomes for patients with a variety of malignancies. However, most patients either do not initially respond or do not exhibit durable responses due to primary or adaptive/acquired immune resistance mechanisms of the tumor microenvironment. These suppressive programs are myriad, different between patients with ostensibly the same cancer type, and can harness multiple cell types to reinforce their stability. Consequently, the overall benefit of monotherapies remains limited. Cutting-edge technologies now allow for extensive tumor profiling, which can be used to define tumor cell intrinsic and extrinsic pathways of primary and/or acquired immune resistance, herein referred to as features or feature sets of immune resistance to current therapies. We propose that cancers can be characterized by immune resistance archetypes, comprised of five feature sets encompassing known immune resistance mechanisms. Archetypes of resistance may inform new therapeutic strategies that concurrently address multiple cell axes and/or suppressive mechanisms, and clinicians may consequently be able to prioritize targeted therapy combinations for individual patients to improve overall efficacy and outcomes.

[18F]FluorThanatrace ([18F]FTT) PET Imaging of PARP-Inhibitor Drug-Target Engagement as a Biomarker of Response in Ovarian Cancer, a Pilot Study.

PURPOSE: PARP inhibitors have become the standard-of-care treatment for homologous recombination deficient (HRD) high-grade serous ovarian cancer (HGSOC). However, not all HRD tumors respond to PARPi. Biomarkers to predict response are needed. [18F]FluorThanatrace ([18F]FTT) is a PARPi-analog PET radiotracer that noninvasively measures PARP-1 expression. Herein, we evaluate [18F]FTT as a biomarker to predict response to PARPi in patient-derived xenograft (PDX) models and subjects with HRD HGSOC. EXPERIMENTAL DESIGN: In PDX models, [18F]FTT-PET was performed before and after PARPi (olaparib), ataxia-telangiectasia inhibitor (ATRi), or both (PARPi-ATRi). Changes in [18F]FTT were correlated with tumor volume changes. Subjects were imaged with [18F]FTT-PET at baseline and after ∼1 week of PARPi. Changes in [18F]FTT-PET uptake were compared with changes in tumor size (RECISTv1.1), CA-125, and progression-free survival (PFS). RESULTS: A decrease in [18F]FTT tumor uptake after PARPi correlated with response to PARPi, or PARPi-ATRi treatment in PARPi-resistant PDX models (r = 0.77-0.81). In subjects (n = 11), percent difference in [18F]FTT-PET after ∼7 days of PARPi compared with baseline correlated with best RECIST response (P = 0.01), best CA-125 response (P = 0.033), and PFS (P = 0.027). All subjects with >50% reduction in [18F]FTT uptake had >6-month PFS and >50% reduction in CA-125. Utilizing only baseline [18F]FTT uptake did not predict such responses. CONCLUSIONS: The decline in [18F]FTT uptake shortly after PARPi initiation provides a measure of drug-target engagement and shows promise as a biomarker to guide PARPi therapies in this pilot study. These results support additional preclinical mechanistic and clinical studies in subjects receiving PARPi ± combination therapy. See related commentary by Liu and Zamarin, p. 1384.

Pre-clinical investigation of astatine-211-parthanatine for high-risk neuroblastoma.

Astatine-211-parthanatine ([211At]PTT) is an alpha-emitting radiopharmaceutical therapeutic that targets poly(adenosine-diphosphate-ribose) polymerase 1 (PARP1) in cancer cells. High-risk neuroblastomas exhibit among the highest PARP1 expression across solid tumors. In this study, we evaluated the efficacy of [211At]PTT using 11 patient-derived xenograft (PDX) mouse models of high-risk neuroblastoma, and assessed hematological and marrow toxicity in a CB57/BL6 healthy mouse model. We observed broad efficacy in PDX models treated with [211At]PTT at the maximum tolerated dose (MTD 36 MBq/kg/fraction x4) administered as a fractionated regimen. For the MTD, complete tumor response was observed in 81.8% (18 of 22) of tumors and the median event free survival was 72 days with 30% (6/20) of mice showing no measurable tumor >95 days. Reversible hematological and marrow toxicity was observed 72 hours post-treatment at the MTD, however full recovery was evident by 4 weeks post-therapy. These data support clinical development of [211At]PTT for high-risk neuroblastoma.

Cryopreserved PM21-Particle-Expanded Natural Killer Cells Maintain Cytotoxicity and Effector Functions In Vitro and In Vivo.

There is a great interest in developing natural killer (NK) cells as adoptive cancer immunotherapy. For off-the-shelf approaches and to conduct multicenter clinical trials, cryopreserved NK cells are the preferred product. However, recent studies reported that cryopreservation of NK cells results in loss of cell motility and, as a consequence, cytotoxicity which limits the clinical utility of such products. This study assessed the impact of cryopreservation on the recovery and function of PM21-particle expanded NK cells (PM21-NK cells) as well as their antitumor activity in vitro using 2D and 3D cancer models and in vivo in ovarian cancer models, including patient-derived xenografts (PDX). Viable PM21-NK cells were consistently recovered from cryopreservation and overnight rest with a mean recovery of 73 ± 22% (N = 19). Thawed and rested NK cells maintained the expression of activating receptors when compared to expansion-matched fresh NK cells. Cryopreserved NK cells that were thawed and rested showed no decrease in cytotoxicity when co-incubated with tumor cells at varying effector-to-target (NK:T) ratios compared to expansion-matched fresh NK cells. Moreover, no differences in cytotoxicity were observed between expansion-matched cryopreserved and fresh NK cells in 3D models of tumor killing. These were analyzed by kinetic, live-cell imaging assays co-incubating NK cells with tumor spheroids. When exposed to tumor cells, or upon cytokine stimulation, cryopreserved NK cells that were thawed and rested showed no significant differences in surface expression of degranulation marker CD107a or intracellular expression of TNFα and IFNγ. In vivo antitumor activity was also assessed by measuring the extension of survival of SKOV-3-bearing NSG mice treated with fresh vs. cryopreserved NK cells. Cryopreserved NK cells caused a statistically significant survival extension of SKOV-3-bearing NSG mice that was comparable to that observed with fresh NK cells. Additionally, treatment of NSG mice bearing PDX tumor with cryopreserved PM21-NK cells resulted in nearly doubling of survival compared to untreated mice. These data suggest that PM21-NK cells can be cryopreserved and recovered efficiently without appreciable loss of viability or activity while retaining effector function both in vitro and in vivo. These findings support the use of cryopreserved PM21-NK cells as a cancer immunotherapy treatment.

Induction of pancreatitis in mice with susceptibility to pancreatic cancer.

Chronic inflammation is known to be associated with pancreatic cancer, however a complete picture regarding how these pathologies intersect is still being characterized. In vivo model systems are critical for the study of mechanisms underlying how inflammation accelerates neoplasia. Repeat injection of cerulein, a cholecystokinin (CCK) analog, is widely used to experimentally induce acute and chronic pancreatitis in vivo. Chronic cerulein administration into genetically engineered mouse models (GEMMs) with predisposition to pancreatic cancer can induce a pro-inflammatory immune response, pancreatic acinar cell damage, pancreatic stellate cell activation, and accelerate the onset of neoplasia. Here we provide a detailed protocol and insights into using cerulein to induce pancreatitis in GEMMs, and methods to experimentally assess inflammation and pancreatic neoplasia.

Humanized Patient-Derived Xenograft Models of Ovarian Cancer.

In vivo modeling of cancer is a critical step in testing novel therapeutic strategies to advance patient care. Here we describe how to develop a humanized patient-derived xenograft (PDX) model of ovarian cancer that uses orthotopically transplanted patient ovarian tumors with autologous transfer of expanded tumor infiltrating T cells (TILs) as a model that can be utilized to test immunomodulating therapeutics in vivo.

Differential Expression of Polyamine Pathways in Human Pancreatic Tumor Progression and Effects of Polyamine Blockade on Tumor Microenvironment.

Pancreatic cancer is the fourth leading cause of cancer death. Existing therapies only moderately improve pancreatic ductal adenocarcinoma (PDAC) patient prognosis. The present study investigates the importance of the polyamine metabolism in the pancreatic tumor microenvironment. Relative mRNA expression analysis identified differential expression of polyamine biosynthesis, homeostasis, and transport mediators in both pancreatic epithelial and stromal cells from low-grade pancreatic intraepithelial neoplasia (PanIN-1) or primary PDAC patient samples. We found dysregulated mRNA levels that encode for proteins associated with the polyamine pathway of PDAC tumors compared to early lesions. Next, bioinformatic databases were used to assess expression of select genes involved in polyamine metabolism and their impact on patient survival. Higher expression of pro-polyamine genes was associated with poor patient prognosis, supporting the use of a polyamine blockade therapy (PBT) strategy for inhibiting pancreatic tumor progression. Moreover, PBT treatment of syngeneic mice injected intra-pancreatic with PAN 02 tumor cells resulted in increased survival and decreased tumor weights of PDAC-bearing mice. Histological assessment of PBT-treated tumors revealed macrophage presence and significantly increased expression of CD86, a T cell co-stimulatory marker. Collectively, therapies which target polyamine metabolism can be used to disrupt tumor progression, modulate tumor microenvironment, and extend overall survival.

DFMO Improves Survival and Increases Immune Cell Infiltration in Association with MYC Downregulation in the Pancreatic Tumor Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor five-year survival rate of less than 10%. Immune suppression along with chemoresistance are obstacles for PDAC therapeutic treatment. Innate immune cells, such as tumor-associated macrophages, are recruited to the inflammatory environment of PDAC and adversely suppress cytotoxic T lymphocytes. KRAS and MYC are important oncogenes associated with immune suppression and pose a challenge to successful therapies. Here, we targeted KRAS, through inhibition of downstream c-RAF with GW5074, and MYC expression via difluoromethylornithine (DFMO). DFMO alone and with GW5074 reduced in vitro PDAC cell viability. Both DFMO and GW5074 showed efficacy in reducing in vivo PDAC growth in an immunocompromised model. Results in immunocompetent syngeneic tumor-bearing mice showed that DFMO and combination treatment markedly decreased tumor size, but only DFMO increased survival in mice. To further investigate, immunohistochemical staining showed DFMO diminished MYC expression and increased tumor infiltration of macrophages, CD86+ cells, CD4+ and CD8+ T lymphocytes. GW5074 was not as effective in modulating the tumor infiltration of total CD3+ lymphocytes or tumor progression and maintained MYC expression. Collectively, this study highlights that in contrast to GW5074, the inhibition of MYC through DFMO may be an effective treatment modality to modulate PDAC immunosuppression.

PARP Theranostic Auger Emitters Are Cytotoxic in BRCA Mutant Ovarian Cancer and Viable Tumors from Ovarian Cancer Patients Enable Ex-Vivo Screening of Tumor Response.

Theranostics are emerging as a pillar of cancer therapy that enable the use of single molecule constructs for diagnostic and therapeutic application. As poly adenosine diphosphate (ADP)-ribose polymerase 1 (PARP-1) is overexpressed in various cancer types, and is localized to the nucleus, PARP-1 can be safely targeted with Auger emitters to induce DNA damage in tumors. Here, we investigated a radioiodinated PARP inhibitor, [125I]KX1, and show drug target specific DNA damage and subsequent killing of BRCA1 and non-BRCA mutant ovarian cancer cells at sub-pharmacological concentrations several orders of magnitude lower than traditional PARP inhibitors. Furthermore, we demonstrated that viable tumor tissue from ovarian cancer patients can be used to screen tumor radiosensitivity ex-vivo, enabling the direct assessment of therapeutic efficacy. Finally, we showed tumors can be imaged by single-photon computed tomography (SPECT) with PARP theranostic, [123I]KX1, in a human ovarian cancer xenograft mouse model. These data support the utility of PARP-1 targeted radiopharmaceutical therapy as a theranostic option for PARP-1 overexpressing ovarian cancers.

Identification of a novel IL-5 signaling pathway in chronic pancreatitis and crosstalk with pancreatic tumor cells.

BACKGROUND: While inflammation is associated with pancreatic cancer, the underlying mechanisms leading to cancer initiation are still being delineated. Eosinophils may promote or inhibit tumor growth, although the specific role in pancreatic cancer has yet to be determined. Eosinophil-supporting cytokine interleukin-5 and receptor are likely to have a role, but the significance in the pancreatic cancer microenvironment is unknown. METHODS: Genetically engineered Akt1Myr/KRasG12D and KRasG12D mice were used to model changes induced by chronic inflammation. Tissue samples were collected to analyze the tumor microenvironment and infiltration of immune cells, whereas serum was collected to analyze cytokine and amylase activity in the inflammatory model. The expression of IL-5R and the effects of IL-5 were analyzed in human and murine tumor cells. RESULTS: Compound Akt1Myr/KRasG12D mice, compared to single KRasG12D or Akt1Myr mice, exhibited increased tissue damage after repeat inductions of inflammation, and had accelerated tumor development and metastasis. M2 macrophages and newly identified eosinophils co-localized with fibrotic regions rather than infiltrating into tumors, consistent with immune cell privilege. The majority of eosinophils found in the pancreas of Akt1Myr/KRasG12D mice with chronic inflammation lacked the cytotoxic NKG2D marker. IL-5 expression was upregulated in pancreatic cells in response to inflammation, and then diminished in advanced lesions. Although not previously described in pancreatic tumors, IL-5Rα was increased during mouse pancreatic tumor progression and expressed in human pancreatic ductal adenocarcinomas (7 of 7 by immunohistochemistry). IL-5 stimulated tumor cell migration and activation through STAT5 signaling, thereby suggesting an unreported tumor-promoting role for IL-5Rα in pancreatic cancer. CONCLUSIONS: Chronic inflammation induces increased pancreatic cancer progression and immune cells such as eosinophils are attracted to areas of fibrosis. Results suggest that IL-5 in the pancreatic compartment stimulates increased IL-5Rα on ductal tumor cells to increase pancreatic tumor motility. Collectively, IL-5/IL-5Rα signaling in the mouse and human pancreatic tumors microenvironment is a novel mechanism to facilitate tumor progression. Additional file 1: Video Abstract.

An autologous humanized patient-derived-xenograft platform to evaluate immunotherapy in ovarian cancer.

OBJECTIVE: The aim of this study was to "humanize" ovarian cancer patient-derived xenograft (PDX) models by autologous transfer of patient-matched tumor infiltrating lymphocytes (TILs) to evaluate immunotherapies. METHODS: Orthotopic high-grade serous ovarian cancer (HGSOC) PDX models were established from three patient donors. Models were molecularly and histologically validated by immunohistochemistry. TILs were expanded from donor tumors using a rapid expansion protocol. Ex vivo TIL and tumor co-cultures were performed to validate TIL reactivity against patient-matched autologous tumor cells. Expression of TIL activation markers and cytokine secretion was quantitated by flow cytometry and ELISA. As proof of concept, the efficacy of anti-PD-1 monotherapy was tested in autologous TIL/tumor HGSOC PDX models. RESULTS: Evaluation of T-cell activation in autologous TIL/tumor co-cultures resulted in an increase in HLA-dependent IFNγ production and T-cell activation. In response to increased IFNγ production, tumor cell expression of PD-L1 was increased. Addition of anti-PD-1 antibody to TIL/tumor co-cultures increased autologous tumor lysis in a CCNE1 amplified model. Orthotopic HGSOC PDX models from parallel patient-matched tumors maintained their original morphology and molecular marker profile. Autologous tumor-reactive TIL administration in patient-matched PDX models resulted in reduced tumor burden and increased survival, in groups that also received anti-PD-1 therapy. CONCLUSIONS: This study validates a novel, clinically relevant model system for in vivo testing of immunomodulating therapeutic strategies for ovarian cancer, and provides a unique platform for assessing patient-specific T-cell response to immunotherapy.

CAR T Cells Targeting MISIIR for the Treatment of Ovarian Cancer and Other Gynecologic Malignancies.

The prognosis of patients diagnosed with advanced ovarian or endometrial cancer remains poor, and effective therapeutic strategies are limited. The Müllerian inhibiting substance type 2 receptor (MISIIR) is a transforming growth factor ß (TGF-ß) receptor family member, overexpressed by most ovarian and endometrial cancers while absent in most normal tissues. Restricted tissue expression, coupled with an understanding that MISIIR ligation transmits apoptotic signals to cancer cells, makes MISIIR an attractive target for tumor-directed therapeutics. However, the development of clinical MISIIR-targeted agents has been challenging. Prompted by the responses achieved in patients with blood malignancies using chimeric antigen receptor (CAR) T cell therapy, we hypothesized that MISIIR targeting may be achieved using a CAR T cell approach. Herein, we describe the development and evaluation of a CAR that targets MISIIR. T cells expressing the MISIIR-specific CAR demonstrated antigen-specific reactivity in vitro and eliminated MISIIR-overexpressing tumors in vivo. MISIIR CAR T cells also recognized a panel of human ovarian and endometrial cancer cell lines, and they lysed a battery of patient-derived tumor specimens in vitro, without mediating cytotoxicity of a panel of normal primary human cells. In conclusion, these results indicate that MISIIR targeting for the treatment of ovarian cancer and other gynecologic malignancies is achievable using CAR technology.

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

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.

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.

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.