Hypophosphatemia Due to Increased Effector Cell Metabolic Activity Is Associated with Neurotoxicity Symptoms in CD19-Targeted CAR T-cell Therapy.

A major complication of chimeric antigen receptor (CAR) T-cell therapy is immune effector cell-associated neurotoxicity syndrome (ICANS), which presents as aphasia, confusion, weakness, somnolence, seizures, and coma. This is similar to the neurologic manifestations of hypophosphatemia, which can result from sudden increases in metabolic demand for phosphorylated intermediates (e.g., refeeding syndrome and sepsis). Given these similarities, we investigated whether CAR T-cell effector metabolic activity is associated with increased extracellular phosphate consumption and a possible association between hypophosphatemia and ICANS. In vitro 4-1BB and CD28 CD19-targeted CAR T-cell effector activity was found to be associated with increased consumption of media phosphorus, which was temporally associated with increased single-cell effector secretomic activity and increased phosphorus-dependent metabolic demand of the CAR T cells. A clinical cohort of 77 patients treated with CD19-targeted CAR T-cell therapy demonstrated a significant anticorrelation between serum phosphorus and ICANS incidence and severity, with earlier onset of hypophosphatemia after CAR T-cell infusion more likely to result in neurotoxicity. These results imply phosphorous level monitoring could alert to the development of ICANS in clinical scenarios. See related Spotlight by Tobin et al., p. 1422.

89Zr-ImmunoPET Shows Therapeutic Efficacy of Anti-CD20-IFNα Fusion Protein in a Murine B-cell Lymphoma Model.

Antibody-mediated tumor delivery of cytokines can overcome limitations of systemic administration (toxicity, short half-lives). Previous work showed improved antitumor potency of anti-CD20-IFNα fusion proteins in preclinical mouse models of B-cell lymphoma. Although tumor targeting is mediated by the antibody part of the fusion protein, the cytokine component might strongly influence biodistribution and pharmacokinetics, as a result of its affinity, size, valency, and receptor distribution. Here, we used immunoPET to study the in vivo biodistribution and tumor targeting of the anti-CD20 rituximab-murine IFNα1 fusion protein (Rit-mIFNα) and compared it with the parental mAb (rituximab, Rit). Rit-mIFNα and Rit were radiolabeled with zirconium-89 (89Zr, t1/2 78.4 hours) and injected into C3H mice bearing syngeneic B-cell lymphomas (38C13-hCD20). Dynamic [(2 hours post injection (p.i.)] and static (4, 24, and 72 hours) PET scans were acquired. Ex vivo biodistribution was performed after the final scan. Both 89Zr-Rit-mIFNα and 89Zr-Rit specifically target hCD20-expressing B-cell lymphoma in vivo. 89Zr-Rit-mIFNα showed specific uptake in tumors (7.6 ± 1.0 %ID/g at 75 hours p.i.), which was significantly lower than 89Zr-Rit (38.4 ± 9.9 %ID/g, P < 0.0001). ImmunoPET studies also revealed differences in the biodistribution, 89Zr-Rit-mIFNα showed rapid blood clearance and high accumulation in the liver compared with 89Zr-Rit. Importantly, immunoPET clearly revealed a therapeutic effect of the single 89Zr-Rit-mIFNα dose, resulting in smaller tumors and fewer lymph node metastases compared with mice receiving 89Zr-Rit. Mice receiving 89Zr-Rit-mIFNα had enlarged spleens, suggesting that systemic immune activation contributes to therapeutic efficacy in addition to the direct antitumoral activity of IFNα. In conclusion, immunoPET allows the noninvasive tracking and quantification of the antibody-cytokine fusion protein and helps understand the in vivo behavior and therapeutic efficacy.

An HK2 Antisense Oligonucleotide Induces Synthetic Lethality in HK1-HK2+ Multiple Myeloma.

Although the majority of adult tissues express only hexokinase 1 (HK1) for glycolysis, most cancers express hexokinase 2 (HK2) and many coexpress HK1 and HK2. In contrast to HK1+HK2+ cancers, HK1-HK2+ cancer subsets are sensitive to cytostasis induced by HK2shRNA knockdown and are also sensitive to synthetic lethality in response to the combination of HK2shRNA knockdown, an oxidative phosphorylation (OXPHOS) inhibitor diphenyleneiodonium (DPI), and a fatty acid oxidation (FAO) inhibitor perhexiline (PER). The majority of human multiple myeloma cell lines are HK1-HK2+. Here we describe an antisense oligonucleotide (ASO) directed against human HK2 (HK2-ASO1), which suppressed HK2 expression in human multiple myeloma cell cultures and human multiple myeloma mouse xenograft models. The HK2-ASO1/DPI/PER triple-combination achieved synthetic lethality in multiple myeloma cells in culture and prevented HK1-HK2+ multiple myeloma tumor xenograft progression. DPI was replaceable by the FDA-approved OXPHOS inhibitor metformin (MET), both for synthetic lethality in culture and for inhibition of tumor xenograft progression. In addition, we used an ASO targeting murine HK2 (mHK2-ASO1) to validate the safety of mHK2-ASO1/MET/PER combination therapy in mice bearing murine multiple myeloma tumors. HK2-ASO1 is the first agent that shows selective HK2 inhibition and therapeutic efficacy in cell culture and in animal models, supporting clinical development of this synthetically lethal combination as a therapy for HK1-HK2+ multiple myeloma. SIGNIFICANCE: A first-in-class HK2 antisense oligonucleotide suppresses HK2 expression in cell culture and in in vivo, presenting an effective, tolerated combination therapy for preventing progression of HK1-HK2+ multiple myeloma tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2748/F1.large.jpg.

18F-labeled anti-human CD20 cys-diabody for same-day immunoPET in a model of aggressive B cell lymphoma in human CD20 transgenic mice.

PURPOSE: Metabolic imaging using [18F]FDG is the current standard for clinical PET; however, some malignancies (e.g., indolent lymphomas) show low avidity for FDG. The majority of B cell lymphomas express CD20, making it a valuable target both for antibody-based therapy and imaging. We previously developed PET tracers based on the humanised anti-CD20 antibody obinutuzumab (GA101). Preclinical studies showed that the smallest bivalent fragment, the cys-diabody (GAcDb, 54.5 kDa) with a peak uptake at 1-2 h post-injection and a biological half-life of 2-5 h, is compatible with short-lived positron emitters such as fluorine-18 (18F, t1/2 110 min), enabling same-day imaging. METHODS: GAcDb was radiolabeled using amine-reactive N-succinimidyl 4-[18F]-fluorobenzoate ([18F]SFB), or thiol-reactive N-[2-(4-[18F]-fluorobenzamido)ethyl]maleimide ([18F]FBEM) for site-specific conjugation to C-terminal cysteine residues. Both tracers were used for immunoPET imaging of the B cell compartment in human CD20 transgenic mice (hCD20TM). [18F]FB-GAcDb immunoPET was further evaluated in a disseminated lymphoma (A20-hCD20) syngeneic for hCD20TM and compared to [18F]FDG PET. Tracer uptake was confirmed by ex vivo biodistribution. RESULTS: The GAcDb was successfully 18F-radiolabeled using two different conjugation methods resulting in similar specific activities and without impairing immunoreactivity. Both tracers ([18F]FB-GAcDb and [18F]FBEM-GAcDb) specifically target human CD20-expressing B cells in transgenic mice. Fast blood clearance results in high contrast PET images as early as 1 h post injection enabling same-day imaging. [18F]FB-GAcDb immunoPET detects disseminated lymphoma disease in the context of normal tissue expression of hCD20, with comparable sensitivity as [18F]FDG PET but with added specificity for the therapeutic target. CONCLUSIONS: [18F]FB-GAcDb and [18F]FBEM-GAcDb could monitor normal B cells and B cell malignancies non-invasively and quantitatively in vivo. In contrast to [18F]FDG PET, immunoPET provides not only information about the extent of disease but also about presence and localisation of the therapeutic target.

Activity of Anti-CD19 Chimeric Antigen Receptor T Cells Against B Cell Lymphoma Is Enhanced by Antibody-Targeted Interferon-Alpha.

An important emerging form of immunotherapy targeting B cell malignancies is chimeric antigen receptor (CAR) T cell therapy. Despite encouraging response rates of anti-CD19 CAR T cell therapy in B cell lymphomas, limited durability of response necessitates further study to potentiate CAR T cell efficacy. Antibody-targeted interferon (IFN) therapy is a novel approach in immunotherapy. Given the ability of IFNs to promote T cell activation and survival, target cell recognition, and cytotoxicity, we asked whether antibody-targeted IFN could enhance the antitumor effects of anti-CD19 CAR T cells. We produced an anti-CD20-IFN fusion protein containing the potent type 1 IFN isoform alpha14 (α14), and demonstrated its ability to suppress proliferation and induce apoptosis of human B cell lymphomas. Indeed, with the combination of anti-CD20-hIFNα14 and CAR T cells, we found enhanced cell killing among B cell lymphoma lines. Importantly, for all cell lines pretreated with anti-CD20-hIFNα14, the subsequent cytokine production by CAR T cells was markedly increased regardless of the degree of cell killing. Thus, several activities of CD19 CAR T cells were enhanced in the presence of anti-CD20-hIFNα14. These data suggest that antibody-targeted IFN may be an important novel approach to improving the efficacy of CAR T cell therapy.

Anti-CD20-interferon-ß fusion protein therapy of murine B-cell lymphomas.

Type I interferons (IFNα/ß) are cytokines with a broad spectrum of antitumor activities including antiproliferative, proapoptotic, and immunostimulatory effects, and are potentially useful in the treatment of B-cell malignancies and other cancers. To improve antitumor potency and diminish the systemic side effects of IFN, we recently developed anti-CD20-IFNα fusion proteins with in vitro and in vivo efficacy against both mouse and human lymphomas expressing CD20. As IFNß binds more tightly to the IFNα/ß receptor (IFNAR) and has more potent antitumor activities, we have now constructed an anti-CD20 fusion protein with murine IFNß (mIFNß). Anti-CD20-mIFNß was more potent than recombinant mIFNß and anti-CD20-mIFNα in inhibiting the proliferation of a mouse B-cell lymphoma expressing human CD20 (38C13-huCD20). Growth inhibition was accompanied by caspase-independent apoptosis and DNA fragmentation. The efficacy of anti-CD20-mIFNß required the physical linkage of mIFNß to anti-CD20 antibody. Importantly, anti-CD20-mIFNß was active against tumor cells expressing low levels of IFNAR (38C13-huCD20 IFNAR). In vivo, established 38C13-huCD20 tumors were largely insensitive to rituximab or a nontargeted mIFNß fusion protein, yet treatment with anti-CD20-mIFNß eradicated 83% of tumors. Anti-CD20-mIFNß was also more potent in vivo against 38C13-huCD20 than anti-CD20-mIFNα, curing 75% versus 25% of tumors (P=0.001). Importantly, although anti-CD20-mIFNα could not eradicate 38C13-huCD20 IFNAR tumors, anti-CD20-mIFNß treatment prolonged survival (P=0.0003), and some animals remained tumor-free. Thus, antibody fusion proteins targeting mIFNß to tumors show promise as therapeutic agents, especially for use against tumors resistant to the effects of mIFNα.

A phase I dose-finding trial of recombinant interleukin-21 and rituximab in relapsed and refractory low grade B-cell lymphoproliferative disorders.

PURPOSE: We conducted a phase I study to determine the safety, maximum-tolerated dose (MTD), and efficacy of weekly bolus recombinant human interleukin-21 (rIL-21) plus rituximab in patients with indolent B-cell malignancies. EXPERIMENTAL DESIGN: One week after a lead-in rituximab dose, cohorts of three patients were treated with 30, 100, or 150 µg/kg rIL-21 weekly for four weeks, concurrent with four weekly doses of rituximab. Patients with stable disease or better were eligible for a second course of therapy. RESULTS: Twenty-one patients with relapsed small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL, n = 11), follicular lymphoma (n = 9), or marginal zone lymphoma (n = 1) were enrolled, with 19 completing at least one course of therapy. The MTD for rIL-21 was 100 µg/kg, based on observed toxicities including nausea, vomiting, diarrhea, hypotension, edema, and hypophosphatemia. Clinical responses were seen in 8 of 19 evaluable patients (42%; 3 CR/CRu, 5 PR), with 4 of longer duration than the patient's previous response to rituximab-based treatment (median 9 months vs. 3 months). CONCLUSIONS: Outpatient therapy of indolent B-cell malignancies with rituximab and weekly rIL-21 was well tolerated and clinically active, with durable complete remissions in a small subset of patients. Additional studies of rIL-21 and anti-CD20 antibodies in lymphoma and SLL/CLL are warranted.

Combination of cyclophosphamide, rituximab, and intratumoral CpG oligodeoxynucleotide successfully eradicates established B cell lymphoma.

Rituximab plus chemotherapy is standard therapy for patients with non-Hodgkin B cell lymphoma, but often complete response or cure is not achieved. Toll-like receptor 9 agonist CpG oligodeoxynucleotides (CpG) can improve antibody-dependent cellular cytotoxicity and adaptive antitumor immune responses. Using a syngeneic murine B cell lymphoma expressing human CD20 (38C13-huCD20), we previously demonstrated that rituximab plus intratumoral CpG, but not systemic CpG, could eradicate up to half of 7-day established 38C13-huCD20 tumors. However, larger 10-day established tumors could not be cured with this regimen. We thus hypothesized that cytoreduction with cyclophosphamide (Cy) before immunotherapy might permit eradication of these more advanced tumor burdens. Pretreatment with Cy resulted in tumor eradication from 83% of animals treated with rituximab/CpG, whereas Cy/CpG or Cy/rituximab treatments only cured 30% or 17%, respectively (P<0.005). Tumor eradication depended on natural killer cells, but not T cells, macrophages, or complement. Only mice treated with Cy/rituximab/CpG partially resisted rechallenge with tumor cells. Foxp3 Treg and CD11bGr1 myeloid suppressor cells persisted within lymphoid organs after therapy, possibly influencing the ability to establish adaptive tumor immunity. In conclusion, cytoreduction with Cy permitted the cure of large, established lymphomas not otherwise responsive to rituximab plus intratumoral CpG immunotherapy.

Programmed death ligand 1 is expressed by non-hodgkin lymphomas and inhibits the activity of tumor-associated T cells.

PURPOSE: Programmed death ligand 1 (PD-L1) is expressed on antigen-presenting cells and inhibits activation of T cells through its receptor PD-1. PD-L1 is aberrantly expressed on some epithelial malignancies and Hodgkin lymphomas and may prevent effective host antitumor immunity. The role of PD-L1 in non-Hodgkin lymphomas (NHL) is not well characterized. EXPERIMENTAL DESIGN: PD-L1 expression was analyzed in cell lines and lymphoma specimens by using flow cytometry and immunohistochemistry. Functional activity of PD-L1 was studied by incubating irradiated lymphoma cells with allogeneic T cells with or without anti-PD-L1 blocking antibody; T-cell proliferation and IFN-γ secretion served as measures of T-cell activation. Similar experiments were conducted using cultures of primary lymphoma specimens containing host T cells. RESULTS: PD-L1 was expressed uniformly by anaplastic large cell lymphoma (ALCL) cell lines, but rarely in B-cell NHL, confined to a subset of diffuse large B-cell lymphomas (DLBCL) with activated B-cell features (3 of 28 cell lines and 24% of primary DLBCL). Anti-PD-L1 blocking antibody boosted proliferation and IFN-γ secretion by allogeneic T cells responding to ALCL and DLBCL cells. In autologous cultures of primary ALCL and DLBCL, PD-L1 blockade enhanced secretion of inflammatory cytokines IFN-γ, granulocyte macrophage colony-stimulating factor, interleukin (IL)-1, IL-6, IL-8, IL-13, TNF-α, and macrophage inflammatory protein-1α. In establishing cell lines from an aggressive PD-L1(+) mature B-cell lymphoma, we also noted that PD-L1 expression could be lost under certain in vitro culture conditions. CONCLUSIONS: PD-L1 may thwart effective antitumor immune responses and represents an attractive target for lymphoma immunotherapy.

Intratumoral but not systemic delivery of CpG oligodeoxynucleotide augments the efficacy of anti-CD20 monoclonal antibody therapy against B cell lymphoma.

The anti-CD20 monoclonal antibody rituximab (Rituxan) has become a mainstay in the treatment of B cell non-Hodgkin lymphomas. The mechanisms of action for rituximab include antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and apoptosis induction. Combination of anti-CD20 antibodies with immunostimulatory agents may improve their efficacy via enhancement of one or more of these mechanisms. Toll-like receptor 9 agonist CpG oligodeoxynucleotides administered systemically have been studied in clinical trials with and without rituximab. However, recent data suggest that intratumoral (IT) delivery of CpG has advantages in the treatment of tumors. Using a syngeneic murine B cell lymphoma line expressing human CD20, we found that IT, but not systemically administered CpG significantly improved the efficacy of rituximab against 7-day established tumors. Rituximab plus IT CpG could eradicate tumors from 42% of mice, whereas systemically administered CpG, with or without rituximab, did not achieve tumor eradication. Both natural killer cells and complement participated in the cure of tumors by rituximab plus IT CpG, apparently by increasing tumor cell sensitivity to complement and ADCC lysis, and by augmenting the cytotoxicity of ADCC effectors. No role for T cells in mediating tumor eradication was demonstrated in this model. These results suggest that previous clinical trials in B cell lymphoma combining systemic administration of CpG with rituximab may have employed suboptimal routes of CpG delivery. Future trials combining IT CpG with anti-CD20 antibodies or the antibody-mediated targeting of CpG directly to the sites of B cell lymphoma may thus be warranted.

Maleimide conjugation markedly enhances the immunogenicity of both human and murine idiotype-KLH vaccines.

The collection of epitopes present within the variable regions of the tumor-specific clonal immunoglobulin expressed by B cell lymphomas (idiotype, Id) can serve as a target for active immunotherapy. Traditionally, tumor-derived Id protein is chemically conjugated to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde to serve as a therapeutic vaccine. While this approach offered promising results for some patients treated in early clinical trials, glutaraldehyde Id-KLH vaccines have failed to induce immune and clinical responses in many vaccinated subjects. We recently described an alternative conjugation method employing maleimide-sulfhydryl chemistry that significantly increased the therapeutic efficacy of Id-KLH vaccines in three different murine B cell lymphoma models, with protection mediated by either CD8(+) T cells or antibodies. We now define in detail the methods and parameters critical for enhancing the in vivo immunogenicity of human as well as murine Id-KLH conjugate vaccines. Optimal conditions for Id sulfhydryl pre-reduction were determined, and maleimide Id-KLH conjugates maintained stability and potency even after prolonged storage. Field flow fractionation analysis of Id-KLH particle size revealed that maleimide conjugates were far more uniform in size than glutaraldehyde conjugates. Under increasingly stringent conditions, maleimide Id-KLH vaccines maintained superior efficacy over glutaraldehyde Id-KLH in treating established, disseminated murine lymphoma. More importantly, human maleimide Id-KLH conjugates were consistently superior to glutaraldehyde Id-KLH conjugates in inducing Id-specific antibody and T cell responses. The described methods should be easily adaptable to the production of clinical grade vaccines for human trials in B cell malignancies.

Dendritic cells loaded with apoptotic antibody-coated tumor cells provide protective immunity against B-cell lymphoma in vivo.

The in vitro priming of tumor-specific T cells by dendritic cells (DCs) phagocytosing killed tumor cells can be augmented in the presence of antitumor monoclonal antibody (mAb). We investigated whether DCs phagocytosing killed lymphoma cells coated with tumor-specific antibody could elicit antitumor immunity in vivo. Irradiated murine 38C13 lymphoma cells were cocultured with bone marrow-derived DCs in the presence or absence of tumor-specific mAb. Mice vaccinated with DCs cocultured with mAb-coated tumor cells were protected from tumor challenge (60% long-term survival), whereas DCs loaded with tumor cells alone were much less effective. The opsonized whole tumor cell-DC vaccine elicited significantly better tumor protection than a traditional lymphoma idiotype (Id) protein vaccine, and in combination with chemotherapy could eradicate preexisting tumor. Moreover, the DC vaccine protected animals from both wild-type and Id-negative variant tumor cells, indicating that Id is not a major target of the induced tumor immunity. Protection was critically dependent upon CD8(+) T cells, with lesser contribution by CD4(+) T cells. Importantly, opsonized whole tumor cell-DC vaccination did not result in tissue-specific autoimmunity. Since opsonized whole tumor cell-DC and Id vaccines appear to target distinct tumor antigens, optimal antilymphoma immunity might be achieved by combining these approaches.

Effect of inhibition of multiple steps of angiogenesis in syngeneic murine pleural mesothelioma.

BACKGROUND: Angiogenesis is a multistep process in which the endothelial cell plays a pivotal role. We hypothesized that the combination of two antiangiogenic agents with distinct mechanisms of action would more effectively inhibit tumor growth than either agent alone in a murine mesothelioma model. METHODS: A syngeneic murine mesothelioma flank tumor model (AB-12) was established in BALB/c mice. Separate adenovirus vectors expressing the cDNAs for human pigment epithelium-derived factor (AdPEDF) and a soluble form of the human vascular endothelial growth factor receptor-1 (Adsflt-1) were administered intratumorally. End points measured included tumor size, animal survival, and microvessel density using CD31 immunohistochemistry. An orthotopic model of mesothelioma was established by implanting AB-12 cells into the murine pleural cavity. Simultaneously, AdPEDF and Adsflt-1 were instilled intrapleurally and tumor burden and survival were recorded. The development of pulmonary emphysema was also assessed by calculating the mean linear intercept (a measure of interalveolar septal distance) in histologic lung sections from tumor-free mice after vector administration. RESULTS: In the flank tumor model, the combination of AdPEDF and Adsflt-1 inhibited tumor growth, prolonged survival, and decreased microvessel density more profoundly compared with either AdPEDF or Adsflt-1 alone. In the orthotopic model, the combination was also more effective in prolonging survival. Intrapleural AdPEDF or Adsflt-1 did not increase the mean linear intercept compared with controls in tumor-free mice. CONCLUSIONS: In this murine model, inhibiting multiple mechanisms of angiogenesis using two agents is a more effective antineoplastic strategy than using either agent alone. In addition, instillation of antiangiogenic gene transfer vectors into the pleural space does not result in histologic evidence of pulmonary emphysema.

Augmenting major histocompatibility complex class I expression by murine tumors in vivo enhances antitumor immunity induced by an active immunotherapy strategy.

OBJECTIVE: Tumors down-regulate major histocompatibility complex class I expression, escaping recognition by the cellular immune response. We hypothesized that augmentation of tumor cell class I expression by interferon-gamma would enhance the cellular antitumor immune response and cure rate of an active immunotherapy strategy. METHODS: B16.F10 tumor cells were exposed to interferon-gamma in culture, and class I expression was quantified using flow cytometry. Syngeneic mice bearing established tumors were injected with interferon-gamma (5000 U, intraperitoneal), and class I expression was assessed using immunohistochemistry. Tumor-specific cytotoxic T lymphocytes were induced in mice by an intratumoral injection of AdCD40L (5 x 10(10) particles), an adenovirus gene transfer vector-based immunotherapy strategy previously demonstrated to augment cellular antitumor immunity. A conjugate-formation assay and the enzyme-linked immunospot assay were used to evaluate the binding and activation of cytotoxic T lymphocytes, respectively. Interferon-gamma was administered to tumor-bearing mice concomitantly with intratumoral AdCD40L. End points measured included the frequencies of cytotoxic T lymphocytes using the enzyme-linked immunospot assay, tumor size, and mouse survival. The role of class I expression was further evaluated by monoclonal antibody blockade in both in vitro and in vivo experiments. RESULTS: B16.F10 cells exposed to interferon-gamma expressed significantly more class I, both in vitro and in vivo, and were able to bind to and activate cytotoxic T lymphocytes more efficiently than untreated cells. Cytotoxic T-lymphocyte frequencies, tumor regression, and the cure rate induced by AdCD40L were augmented by the addition of a single dose of interferon-gamma in tumor-bearing mice. These in vitro and in vivo effects of interferon-gamma were attenuated by class I monoclonal antibody blockade. CONCLUSIONS: Up-regulation of class I expression using interferon-gamma enhances the cellular antitumor immune response and cure rate of AdCD40L, an active immunotherapy strategy. This approach may be useful for human tumors that lack class I expression.

Cisplatin augments cytotoxic T-lymphocyte-mediated antitumor immunity in poorly immunogenic murine lung cancer.

OBJECTIVE: Many tumors are poorly immunogenic and resistant to cytotoxic T-lymphocyte-mediated cell lysis. Because cisplatin has been demonstrated to increase tumor cell Fas receptor expression, we hypothesized that cisplatin will enhance cytotoxic T-lymphocyte tumor cell killing and augment the antitumor effect of an active immunotherapy strategy in a poorly immunogenic murine lung cancer model. METHODS: Lewis lung carcinoma cells were exposed to cisplatin in vitro, and Fas receptor expression and apoptosis in response to an agonistic anti-Fas antibody were quantified using flow cytometry. Wild-type and Fas ligand-deficient mice bearing Lewis lung carcinoma flank tumors were then treated with intraperitoneal cisplatin as well as an intratumoral injection of an adenovirus gene transfer vector encoding CD40 ligand. End points included tumor size, animal survival, and Fas expression (determined using immunofluorescence). Cytotoxicity assays were performed using splenocytes from adenovirus gene transfer vector encoding CD40 ligand-treated animals as effectors and cisplatin-treated Lewis lung carcinoma cells as targets. RESULTS: Cisplatin induced heightened expression of Fas receptor on Lewis lung carcinoma cells in vitro and in vivo and enhanced apoptosis in cells exposed to an agonistic anti-Fas antibody. In vivo, the combination of 1 dose of intraperitoneal cisplatin and intratumoral adenovirus gene transfer vector encoding CD40 ligand inhibited tumor growth and prolonged survival compared with adenovirus gene transfer vector encoding CD40 ligand alone, resulting in a higher cure rate. This effect was lost in Fas ligand-deficient mice. Splenocytes from adenovirus gene transfer vector encoding CD40 ligand-treated wild-type mice lysed cisplatin-treated Lewis lung carcinoma cells more efficiently than untreated Lewis lung carcinoma cells, an effect lost in splenocytes from Fas ligand-deficient mice. CONCLUSION: Cisplatin augments the antitumor effect of a cytotoxic T-lymphocyte-mediated immunotherapy strategy, resulting in a higher cure rate than seen with immunotherapy alone. This effect is associated with the enhanced ability of cytotoxic T lymphocytes to lyse tumor cells that have been exposed to cisplatin through Fas/Fas ligand interactions.

In vivo gene transfer of pigment epithelium-derived factor inhibits tumor growth in syngeneic murine models of thoracic malignancies.

OBJECTIVE: Pigment epithelium-derived factor is known to be an inhibitor of angiogenesis. We hypothesized that in vivo gene transfer of pigment epithelium-derived factor may inhibit tumor angiogenesis and growth in syngeneic models of thoracic malignancies. METHODS: An adenovirus vector encoding the human pigment epithelium-derived factor cDNA (AdPEDF) was used to transduce human lung cancer cells in vitro. Transgene expression was assessed using Western analysis. Three different murine flank tumors (2 lung, 1 colon) were then established in syngeneic mice and treated intratumorally with phosphate-buffered saline, AdPEDF, or an empty vector (AdNull). Endpoints measured included transgene expression, tumor size, and animal survival, as well as microvessel density within the tumor. Additionally, a murine pulmonary metastasis model was established by intravenous injection of a syngeneic colon adenocarcinoma cell line expressing a marker gene (beta-galactosidase). One day later, treatment (phosphate-buffered saline, AdNull, or AdPEDF) was administered intrapleurally. Tumor burden (gross and histologic inspection, lung weight, and beta-galactosidase expression) was then evaluated 13 days after vector dosing, and survival was recorded. RESULTS: AdPEDF-derived expression of pigment epithelium-derived factor was demonstrated in vitro and in vivo. In syngeneic murine lung cancer flank tumors, intratumoral administration of AdPEDF significantly inhibited tumor growth (P <.01), prolonged mouse survival (P <.01), and decreased microvessel density (P <.01) compared with control groups. In the pulmonary metastasis model, AdPEDF-treated mice exhibited significantly reduced lung lesions, lung weight (P <.0005), beta-galactosidase expression (P <.05), and animal survival was prolonged (P <.05). CONCLUSION: Gene transfer of pigment epithelium-derived factor suppresses tumor vascularization and growth, while prolonging survival in syngeneic murine models of thoracic malignancies.