Cellular Therapy In Combination Research Articles

Direct and antibody-dependent cell-mediated cytotoxicity of head and neck squamous cell carcinoma cells by high-affinity natural killer cells

High affinity natural killer cells (haNKs) are a cell therapy product capable of mediating both direct and antibody-dependent cell-mediated cytotoxicity (ADCC). These cells may be particularly useful in tumors that escape T-cell anti-tumor immunity by harboring antigen processing and presentation defects. Here, we demonstrated that haNKs directly kill both HPV-positive and negative head and neck squamous cell carcinoma cells. Variable tumor cell sensitivity to haNK direct cytotoxicity did not correlated with MHC class I chain-related protein A or B (MICA or MICB) expression. Importantly, haNK killing was significantly enhanced via ADCC mediated by cetuximab or avelumab in cells with higher baseline EGFR or PD-L1 expression, respectively. The ability of IFNγ to induce tumor cell PD-L1 expression correlated with enhanced PD-L1-specific ADCC. IFNγ induced neither tumor cell EGFR expression nor EGFR-specific ADCC. Although a single dose of 8 Gy IR did not appear to directly enhance susceptibility to haNK killing alone, enhanced PD-L1- and EGFR-mediated ADCC after IR correlated with increased PD-L1 and EGFR expression in one of four models. This pre-clinical evidence supports the investigation of haNK cellular therapy in combination with ADCC-mediating mAbs, with or without IR, in the clinical trial setting for patients with advanced HNSCCs. Given the MHC-unrestricted nature of this treatment, it may represent an opportunity to treat patients with non-T-cell inflamed tumors.

Cellular therapy in combination with cytokines improves survival in a xenograft mouse model of ovarian cancer.

Studies have shown enhanced survival of ovarian cancer patients in which the tumors are infiltrated with tumor infiltrating lymphocytes and natural killer cells showing the importance of immune surveillance and recognition in ovarian cancer. Therefore, in this study, we tested cellular immunotherapy and varying combinations of cytokines (IL-2 and/or pegylated-IFNα-2b) in a xenograft mouse model of ovarian cancer. SKOV3-AF2 ovarian cancer cells were injected intra-peritoneally (IP) into athymic nude mice. On day 7 post-tumor cell injection, mice were injected IP with peripheral blood mononuclear cells (PBMC; 5×10(6) PBMC) and cytokine combinations [IL-2±pegylated-IFNα-2b (IFN)]. Cytokine injections were continued weekly for IFN (12,000 U/injection) and thrice weekly for IL-2 (4000 U/injection). Mice were euthanized when they became moribund due to tumor burden at which time tumor and ascitic fluid were measured and collected. Treatment efficacy was measured by improved survival at 8 weeks and overall survival by Kaplan-Meier analysis. We observed that the mice tolerated all treatment combinations without significant weight loss or other apparent illness. Mice receiving PBMC plus IL-2 showed improved median survival (7.3weeks) compared to mice with no treatment (4.2weeks), IL-2 (3.5weeks), PBMC (4.0weeks), or PBMC plus IL-2 and IFN (4.3weeks), although PBMC plus IL-2 was not statistically different than PBMC plus IFN (5.5weeks, p>0.05). We demonstrate that cytokine-stimulated cellular immune therapy with PBMC and IL-2 was well tolerated and resulted in survival advantage compared to untreated controls and other cytokine combinations in the nude-mouse model.

Cellular Therapy, a Novel Treatment Option for Intellectual Disability: A Case Report

Intellectual Disability is a non progressive developmental condition which significantly affects cognition, learning and adaptive behavior of an individual. Currently, there is no treatment available for intellectual disability. However, the therapies available focus mainly on the symptoms of the patient and do not address its core neuropathology. Neurorestorative strategies such as cellular therapy may benefit in these disorders. To study the effect of cellular therapy in intellectual disability, we administered a 13 year old boy with autologous bone marrow mononuclear cells, intrathecally. As a part of the protocol, he was also put on a personalized rehabilitation program. Follow up was done at 3 months and 6 months. No major adverse events were recorded post intervention. In a period of 6 months, he showed improved eye contact, cognition, learning ability, behavior and ability to perform activities of daily living. His score on Functional Independence Measure increased from 67 to 76. On comparing the pre and post PET CT scan, improvement in metabolic activity of hippocampus, left amygdala and cerebellum was recorded. These changes correlated to the functional outcome. These changes suggest that cellular therapy in combination with rehabilitation may repair the neuronal networks in intellectual disability, hence improving processing of information. It was found to be a safe and effective therapeutic modality. Hence, cellular therapy may provide hope for patients and caregivers to improve their quality of life.

A clinical study of autologous bone marrow mononuclear cells for cerebral palsy patients: a new frontier.

Cerebral palsy is a nonprogressive heterogeneous group of neurological disorders with a growing rate of prevalence. Recently, cellular therapy is emerging as a potential novel treatment strategy for cerebral palsy. The various mechanisms by which cellular therapy works include neuroprotection, immunomodulation, neurorestoration, and neurogenesis. We conducted an open label, nonrandomized study on 40 cases of cerebral palsy with an aim of evaluating the benefit of cellular therapy in combination with rehabilitation. These cases were administered autologous bone marrow mononuclear cells intrathecally. The follow-up was carried out at 1 week, 3 months, and 6 months after the intervention. Adverse events of the treatment were also monitored in this duration. Overall, at six months, 95% of patients showed improvements. The study population was further divided into diplegic, quadriplegic, and miscellaneous group of cerebral palsy. On statistical analysis, a significant association was established between the symptomatic improvements and cell therapy in diplegic and quadriplegic cerebral palsy. PET-CT scan done in 6 patients showed metabolic improvements in areas of the brain correlating to clinical improvements. The results of this study demonstrate that cellular therapy may accelerate the development, reduce disability, and improve the quality of life of patients with cerebral palsy.

Abstract 3518: Cellular therapy in combination with cytokines improves survival in a xenograft mouse model of ovarian cancer

Abstract Introduction: Epithelial ovarian cancer is the leading cause of death among gynecologic malignancies, and two-thirds of patients present with stage III and IV disease. Unfortunately, the vast majority of advanced-stage ovarian cancer (OC) patients will relapse, require ongoing treatment, and eventually succumb to chemotherapy-resistant disease. The aim of this study was to test cellular therapy in combination with cytokines (IL-2 and/or pegylated-IFNα-2b) in a xenograft mouse model of OC. Methods: SKOV3-AF2 OC cells (1x106) were injected intraperitoneally (IP) into athymic nude mice (n=40). Day-7 post OC cell injection, mice were IP injected with mononuclear cells (MC; 5x106) and cytokine combinations [IL-2 (4,000 U) ± pegylated-IFNα-2b (IFN; 12,000 U); n=5 per group]. Cytokine injections were continued weekly for IFN and thrice weekly for IL-2. Mice were sacrificed when they became moribund due to tumor burden at which time solid tumor and ascitic fluid was measured and collected. Total RNA was isolated from tumor samples and reverse transcription (RT) reactions were performed. Gene expression of Survivin, DNA mismatch repair homolog (MLH1), cysteine-rich 61 (CCN1), and suppressor of cytokine signaling 1 (SOCS1), which have been shown to play roles in the pathobiology of OC, was analyzed by quantitative (q)RT-PCR using Taqman probes. Results: The in vivo model demonstrated that mice tolerated all the treatment combinations well (PBS, IL-2, IFN, IL-2+IFN, MC, MC+IL-2, MC+IFN, MC+IL-2+IFN) without significant weight loss or other cytotoxicities. Mice receiving MC+IL-2 treatment showed improved survival at 9-weeks post-tumor cell injection (60%, p<0.05) vs. mice without treatment (20%), IL-2 (0%), or MC (20%). There was no significant survival advantage in mice receiving MC+IFN (40%) or MC+IL-2+IFN (20%). Harvested tumors consisted of poorly differentiated surface epithelial carcinoma, growing in solid nests and sheets of large cells with a moderate amount of amphophilic or clear cytoplasm. Tumor cells focally were pleomorphic and multinucleated. Mitoses were frequent with abnormal forms present indicating a high rate of proliferation. We found that all tumors analyzed expressed Survivin, MLH1, CCN1, and SOCS1; however, there was no significant correlation of gene expression level (> 3-fold change) with time to sacrifice, tumor volume, or ascitic fluid production. SOCS1 gene expression, which is known to down-regulate proangiogenic molecules, was up-regulated in IFN treated animals (3-fold increase) but not control animals or animals receiving IL-2. Conclusions: IFNα-2b up-regulates SOCS1 expression in our in vivo mouse model. Cytokine-stimulated cellular immune therapy produces an anti-tumor effect in vivo. This data strongly supports development of cellular therapy (autologous or allogeneic) and is a potentially useful therapeutic strategy for the treatment of OC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3518. doi:1538-7445.AM2012-3518

Abstract 2708: Functional characterization of a fluorescent highly tumorigenic ovarian cancer line to test cellular therapy in experimental models

Abstract Introduction: Because current therapies for ovarian cancer have little impact on the long-term survival, there is a compelling need to develop innovative strategies. Our aim was to functionally characterize a fluorescent highly tumorigenic ovarian cancer line to test cellular therapy in combination with cytokines or chemotherapies in experimental models. Methods: Two fluorescent highly tumorigenic sub-lines (SKOV3-AF1 and SKOV3-AF2) were derived from the SKOV-3 ovarian cancer cell line. Peripheral blood mononuclear cells (PBMC)-mediated cytotoxicity in combination with IL-2 and IFNα-2b of SKOV3-AF1 and -AF2 was assayed by lactate dehydrogenase release. Sensitivity of SKOV3-AF2 cells to polyethylene glycol (PEG)-IFNα-2b or IL-2 was assayed in a xenograph nude mouse model. Histopathology was performed on tumors harvested from mice to determine necrosis and tumor infiltrating lymphocytes in the solid tumors. Results: SKOV3-AF1 and -AF2 sub-lines exhibit increased cytotoxicity mediated by PBMC, IL-2, and IFNα-2b compared to parental SKOV-3 cells. The average cytotoxicity was 59% for both SKOV3-AF1 and -AF2 compared to 28% for SKOV-3 parental cells (p<0.05). SKOV3-AF1 and -AF2 cells are more tumorigenic in vivo compared to SKOV-3 cells as indicated by tumor incidence (SKOV3 66%, SKOV3-AF1 100%, SKOV3-AF2 100%), time to sacrifice, tumor weight, and ascitic fluid production. In addition, the SKOV3-AF2 cells continue to fluoresce when harvested from the mice. SKOV3-AF2 tumor growth was inhibited by PEG-IFNα-2b but not low-dose IL-2. The average tumor weight was 0.96 g in the control treatment group that did not receive PEG-IFNα-2b versus 0.23 g for PEG-IFNα-2b (12,000 U per week); p < 0.05). There was no significant difference in any of the IL-2 treatment groups (0-4,000 U, thrice weekly). Both PEG-IFNα-2b and IL-2 were well tolerated by the mice as demonstrated by no significant weight loss during the treatment. Histopathology revealed that the tumors consisted of poorly differentiated surface epithelial carcinoma and there was no significant difference in the percent necrosis or TIL score in any of the treatment groups. Conclusions: Characterization of SKOV3-AF2 cells revealed that these cells are more susceptible to PBMC-mediated cytotoxicity than SKOV-3 cells, are highly tumorigenic in a xenograph model, and continue to fluoresce when harvested from mice. Our data suggests that the SKOV3-AF2 sub-line will be a useful tool to test cellular therapy for the treatment of ovarian cancer utilizing experimental models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2708. doi:10.1158/1538-7445.AM2011-2708

Functional Characterization of a Fluorescent Highly Tumorigenic Ovarian Cancer Line to Test Cellular Therapy in Experimental Models

The objective of this study was to functionally characterize a fluorescent highly tumorigenic ovarian cancer line to test cellular therapy in combination with cytokines or chemotherapies in experimental models. A fluorescent highly tumorigenic subline (SKOV3-AF2) was derived from the SKOV3 ovarian cancer cell line. Peripheral blood mononuclear cell (PBMC)-mediated cytotoxicity of SKOV3-AF2 in the presence of interleukin 2 (IL-2) and interferon α-2b (IFNα-2b) was assayed by lactate dehydrogenase release. Sensitivity of SKOV3-AF2 cells to polyethylene glycol-IFNα-2b and IL-2 was assayed in a xenograph nude mouse model. Histopathology was performed to determine necrosis and tumor-infiltrating lymphocytes in the solid tumors. Reverse transcriptase-polymerase chain reaction was used for gene expression analyses of E-cadherin and cysteine-rich 61 (CCN1). The SKOV3-AF2 subline exhibits increased cytotoxicity (up to 70%), mediated by PBMCs, IL-2, and IFNα-2b, compared with parental SKOV3-red fluorescent protein (RFP) cells. SKOV3-AF2 cells are more tumorigenic in vivo as indicated by tumor incidence, time to sacrifice, tumor weight, and ascitic fluid production. SKOV3-AF2 tumor growth was inhibited by polyethylene glycol-IFNα-2b but not low-dose IL-2. Histopathology revealed that the tumors consisted of poorly differentiated surface epithelial carcinoma. SKOV3-RFP, and -AF2 cell lines as well as -AF2 tumors expressed E-cadherin. SKOV3-AF2 derived tumors expressed CCN1; however, the SKOV3-RFP and SKOV3-AF2 cell lines did not. Characterization of SKOV3-AF2 cells revealed that it is more susceptible to PBMC-mediated cytotoxicity than SKOV3-RFP and highly tumorigenic in a xenograph model, and AF-2 tumors express genes that promote aggressive behavior. Collectively, our data suggest that the SKOV3-AF2 subline will be a useful tool to test cellular therapy for the treatment of ovarian cancer utilizing experimental models.

Abstract 5622: Development of an experimental model to test cellular therapy in combination with cytokines as a novel treatment for ovarian cancer

Abstract Because current therapies for ovarian cancer have little impact on long-term survival, there is a compelling need to develop innovative treatment strategies. Our aim was to develop in vitro and in vivo models to test cellular therapy in combination with cytokines or chemotherapies to elicit a graft versus tumor response to treat refractory ovarian cancer patients. SKOV-3 ovarian cancer cells, which were transfected with Red Fluorescent Protein (SKOV3-RFP) were passaged through nude mice to create more tumorigenic sub-lines. Two sub-lines were established, SKOV3-AF1 and SKOV3-AF2, which showed increased tumorigenicity compared to parental SKOV3-RFP cells. Both lines resulted in 100% tumor incidence in 6-8 weeks at a dose of 1 × 106 cells compared to 67% tumor incidence at 14 weeks at a dose of 5 × 106 SKOV3-RFP cells. SKOV3-AF1 and -AF2 cells were assayed to determine if peripheral blood mononuclear cells (PBMC) in combination with the cytokines IL-2 and IFNα-2b elicited a cytotoxic effect against the ovarian cancer cells. SKOV3-AF1 and-AF2 sub-lines were highly sensitive to cytotoxicity (up to 70%) mediated by PBMC (20:1 PBMC: SKOV3) in combination with IL-2 (25 ng/ml) and IFNα-2b (500 U/ml). The SKOV3-AF1 and -AF2 cells showed an average of 25% increased cytoxicity compared to parental SKOV3-RFP cells (p<0.05, n=3). Interestingly, SKOV3-AF1 and -AF2 were more resistant to IFNα-2b compared to SKOV3-RFP when cultured in the presence of IFNα-2b alone. In addition, gene expression of E-cadherin and Cysteine-rich 61 (CCN1), which have been shown to play a role in the patho-biology of ovarian cancer, was assessed in SKOV3-RFP, -AF1, and -AF2 cells as well as solid tumors harvested from mice. All SKOV3-derived lines and solid tumor expressed E-cadherin by RT-PCR. None of the SKOV3-derived lines expressed CCN1; however, one of the two SKOV3-AF1 solid tumors tested expressed CCN1. Both SKOV3-AF2 solid tumors evaluated showed weak expression of CCN1. The SKOV3-derived sub-lines that we developed will be a better model to test novel ovarian cancer treatment regimens because these lines were found to be more susceptible to PBMC-mediated cytotoxicity, exhibit increased resistance to IFNα-2b, are more tumorigenic in a xenograph model, and tumors harvested from these lines express genes that make these tumors more aggressive. Collectively, these results suggest that cytokine enhanced cellular therapy may be a viable treatment option for ovarian cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5622.

Molecular characterization of highly tumorigenic cell lines used in a xenograph model to investigate cellular therapy for the treatment of refractory ovarian cancer

e16516 Background: Because current therapies for ovarian cancer (OC) have little impact on the long-term survival, there is a compelling need to develop innovative strategies. Our aim was to characterize OC cell lines which can be utilized to test cellular therapy in combination with cytokines or chemotherapies to elicit a graph versus tumor response to treat refractory OC patients. Methods: SKOV-3-RFP, a red fluorescent protein (RFP) expressing line, was passaged through nude mice to create more tumorigenic lines compared to parental SKOV-3 cells. Three SKOV-3-derived lines were established and characterized for IFNα-2b sensitivity, E-cadherinand CCN1gene expression, and STAT3 activation. The cells were grown in the presence of IFNα-2b to determine proliferative effects. Tumors were harvested when mice became moribund; ascitic fluid (AF) and solid tumor (ST) tissue were snap-frozen. Expression of E-cadherinand CCN1, which have been implicated to play role(s) in OC pathobiology, were analyzed in ST harvested from the mice and from SKOV3-derived lines (SKOV3-RFP, AF1, AF2, and AF3). We investigated the activation status in the SKOV3-derived cells of STAT3, which has been associated with malignant transformation and tumor progression. Results: Parental SKOV3-RFP cells when injected at a dose of 5x106 gave a tumor incidence of 4/6 in 14 weeks. AF1 and AF2, when injected at a dose of 1x106 resulted in 100% tumor incidence in 5-weeks (n = 5). These two lines were also more resistant to IFNα-2b compared to RFP. All SKOV3-derived lines and ST expressed E-cadherin by RT-PCR. None of the SKOV3-derived lines expressed CCN1; however, one of the two AF1 ST tested expressed CCN1. Both AF2 ST evaluated showed weak expression of CCN1. In vitro DNA-binding with electrophoretic mobility shift assay (EMSA), showed AF1 and AF3 cells harbor constitutively-active STAT3, whereas the RFP line does not. Conclusions: The SKOV3-derived lines that we developed will be a better model to test novel OC treatment regiments because these lines exhibit increased resistance to IFNα-2b, are more tumorigenic in a xenograph model, show aberrant STAT3 activation, and tumors harvested from these lines express genes that make these lines more aggressive. No significant financial relationships to disclose.

In VivoAntitumor Effect of Cytotoxic T Lymphocytes Engineered to Produce Interferon-γ by Adenovirus-Mediated Genetic Transduction

Immunotherapy with adoptive transfer of genetically-modified cytotoxic T lymphocytes (CTL) is a promising approach for cancer gene therapy. We developed an adoptive therapy model with murine tumor-specific CTL, to which very efficient (up to 100%) gene introduction was achieved by using recombinant adenoviral vectors. Through a comparative study on the antitumor effects of CTL genetically modified with cytokine genes, transduction with interferon-γ gene resulted in a prominent increase in therapeutic efficacy of CTL in both metastatic and subcutaneous tumor models. Further additive effect was obtained by the adoptive cellular therapy in combination with vaccination of cytokine gene-modified tumor cells. Our findings provide a hopeful strategy of adoptive immunotherapy for human cancers.