Inducible Suicide Gene Research Articles

A double inducible cell ablation system for eliminating senescent astrocytes via apoptosis.

Cell senescence stands as a principal risk factor for various neurodegenerative diseases, with astrocytic senescence emerging as a potentially pivotal player in the pathogenesis of aging and neurodegenerative disorders. Clearing senescent astrocytes holds promise as a potential therapeutic approach for senescence-related diseases. In this study, we designed and constructed two plasmids aimed at inducing apoptosis in senescent astrocytes. This was achieved through the ligation of FKBP (FK506-binding protein) and FRB (FKBP and FKBP rapamycin binding domain) and the formation of caspase8 dimers, thereby achieving the purpose of eliminating senescent astrocytes. The developed vector system demonstrates a specifically capability to induce apoptosis in aging astrocytes, offering a targeted approach to eliminate these cells. The utilization of the double -inducible suicide gene system provides a versatile tool forstimulating cell apoptosis and inhibiting cellular senescence. This system proves valuable in exploring the intrinsic roles and molecular mechanisms of senescent cells in the occurrence and development of aging-related diseases. Ultimately, it offers a potential avenue for developing an efficient treatment system for such conditions.

RapaCaspase-9-based suicide gene applied to the safety of IL-1RAP CAR-T cells.

Even if adoptive cell transfer (ACT) has already shown great clinical efficiency in different types of disease, such as cancer, some adverse events consistently occur, and suicide genes are an interesting system to manage these events. Our team developed a new medical drug candidate, a chimeric antigen receptor (CAR) targeting interleukin-1 receptor accessory protein (IL-1RAP), which needs to be evaluated in clinical trials with a clinically applicable suicide gene system. To prevent side effects and ensure the safety of our candidate, we devised two constructs carrying an inducible suicide gene, RapaCasp9-G or RapaCasp9-A, containing a single-nucleotide polymorphism (rs1052576) affecting the efficiency of endogenous caspase 9. These suicide genes are activated by rapamycin and based on the fusion of human caspase 9 with a modified human FK-binding protein, allowing conditional dimerization. RapaCasp9-G- and RapaCasp9-A-expressing gene-modified T cells (GMTCs) were produced from healthy donors (HDs) and acute myeloid leukemia (AML) donors. The RapaCasp9-G suicide gene demonstrated better efficiency, and we showed its in vitro functionality in different clinically relevant culture conditions. Moreover, as rapamycin is not pharmacologically inert, we also demonstrated its safe use as part of our therapy.

Anthracycline-free tumor elimination in mice leads to\xa0functional and molecular cardiac recovery from cancer-induced alterations in contrast to long-lasting doxorubicin treatment effects

Systemic effects of advanced cancer impact on the heart leading to cardiac atrophy and functional impairment. Using a murine melanoma cancer model (B16F10 melanoma cells stably transduced with a Ganciclovir (GCV)-inducible suicide gene), the present study analysed the recovery potential of cancer-induced cardiomyopathy with or without use of doxorubicin (Dox). After Dox-free tumor elimination and recovery for 70 ± 5 days, cancer-induced morphologic, functional, metabolic and molecular changes were largely reversible in mice previously bearing tumors. Moreover, grip strength and cardiac response to angiotensin II-induced high blood pressure were comparable with healthy control mice. In turn, addition of Dox (12 mg/kg BW) to melanoma-bearing mice reduced survival in the acute phase compared to GCV-alone induced recovery, while long-term effects on cardiac morphologic and functional recovery were similar. However, Dox treatment was associated with permanent changes in the cardiac gene expression pattern, especially the circadian rhythm pathway associated with the DNA damage repair system. Thus, the heart can recover from cancer-induced damage after chemotherapy-free tumor elimination. In contrast, treatment with the cardiotoxic drug Dox induces, besides well-known adverse acute effects, long-term subclinical changes in the heart, especially of circadian clock genes. Since the circadian clock is known to impact on cardiac repair mechanisms, these changes may render the heart more sensitive to additional stress during lifetime, which, at least in part, could contribute to late cardiac toxicity.

Abstract 4069: Selective ablation of solid tumors using a p53-targeted FAST-LNP gene therapy

Abstract While chemotherapy is a key treatment strategy for many solid tumors, it is rarely curative as patients will eventually become resistant. In this study, we sought to develop an effective suicide gene therapy approach for solid tumors that specifically exploits their unique transcriptional activation state. The tumor suppressor p53 is frequently mutated or dysregulated in cancer, and as a result the upstream signaling pathways activating p53 transcription are strongly upregulated. RNA-seq analysis has demonstrated that p53 transcription is significantly upregulated in almost all forms of cancer. Additionally, HCT116 cells lacking functional p53 display a 6-fold increase in p53 promoter activity when compared to its wild type p53 parent cell line. To exploit this, we have developed a Fusogenix FAST-LNP formulation to deliver a p53-driven inducible suicide gene, iCasp9, to solid tumors and destroy them upon activation with small molecule dimerizer, Rapamycin. To establish a proof-of-concept, plasmid encoding iCasp9 and luciferase under control of the p53 promoter was constructed and evaluated in a panel of cancer cell lines. While LNPs administered without Rapamycin or Rapamycin administered alone had no impact on cell viability, we observed greater than 90% apoptotic cell death when both were employed in a wide range of cancer cell lines with p53 deletions or mutations, as measured using cell viability assays, imaging assays, as well as Annexin V and TUNEL flow cytometry. Induction of iCasp9 protein expression and caspase-mediated apoptosis was confirmed using Western blot. No cell death was observed in cells with intact p53 such as human umbilical vein endothelial cells or the fibroblast cell line IMR-90. Next, we assessed the efficacy of FAST-LNPs containing p53-iCasp9 in xenograft PC-3 and H1299 models of human prostate cancer and lung cancer respectively. In some experiments, tumors were implanted subcutaneously in the flanks of 30 mice and allowed to grow to 500 mm3 before treatment by intravenous doses of 100 µg LNP twice per week during continuous low dosing of Rapamycin. We observed a rapid and dramatic reduction in tumor volume averaging 87% over the following 48 hours, with durable response. Tumors in control mice continued to grow exponentially. Overall survival of mice was extended 250% in the PC-3 cohort and 300% in the H1299 cohort. Optimization of number and concentration of LNP doses should allow for long term control of both localized and systemic disease. In conclusion, we describe a novel LNP gene therapy approach for the treatment of cancer with high selectivity for tumors with dysregulated p53 transcriptional activation. This approach has the potential to provide a highly efficacious alternative to current therapies for localized and advanced solid tumors. Citation Format: Douglas Wilson Brown, Arun Raturi, Prakash Bhandari, Deborah Sosnowski, Liliya Grin, Ping Wee, Hector Vega, Jennifer Gyoba, Maryam Hejazi, Jailal Ablack, Matthew Scholz, John D. Lewis. Selective ablation of solid tumors using a p53-targeted FAST-LNP gene therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4069.

Universal Ready-to-Use Immunotherapeutic Approach for the Treatment of Cancer: Expanded and Activated Polyclonal γδ Memory T Cells.

In the last years, important progresses have been registered in the treatment of patients suffering from oncological/haematological malignancies, but more still needs to be done to reduce toxicity and side effects, improve outcome and offer new strategies for relapsed or refractory disease. A remarkable part of these clinical benefits is due to advances in immunotherapy. Here, we investigate the generation of a novel, universal and ready-to-use immunotherapeutic product based on γδ-T lymphocytes. These cells are part of the innate immune system, exerting potent natural cytotoxicity against bacteria, viruses and tumours. This ability, coupled with their negligible alloreactivity, makes them attractive for adoptive immunotherapy approaches. To achieve a cell product suitable for clinical use, we developed a strategy capable to generate polyclonal γδ-T cells with predominant memory-Vδ1 phenotype in good manufacturing practice (GMP) procedures with the additional possibility of gene-modification to improve their anti-tumour activity. Irradiated, engineered artificial antigen-presenting cells (aAPCs) expressing CD86/41BBL/CD40L and the cytomegalovirus (CMV)-antigen-pp65 were used. The presence of CMV-pp65 and CD40L proved to be crucial for expansion of the memory-Vδ1 subpopulation. To allow clinical translation and guarantee patient safety, aAPCs were stably transduced with an inducible suicide gene. Expanded γδ-T cells showed high expression of activation and memory markers, without signs of exhaustion; they maintained polyclonality and potent anti-tumour activity both in vitro (against immortalised and primary blasts) and in in vivo studies without displaying alloreactivity signals. The molecular characterisation (phophoproteomic and gene-expression) of these cell products underlines their unique properties. These cells can further be armed with chimeric antigen receptors (CAR) to improve anti-tumour capacity and persistence. We demonstrate the feasibility of establishing an allogeneic third-party, off-the-shelf and ready-to-use, γδ-T-cell bank. These γδ-T cells may represent an attractive therapeutic option endowed with broad clinical applications, including treatment of viral infections in highly immunocompromised patients, treatment of aggressive malignancies refractory to conventional approaches, bridging therapy to more targeted immunotherapeutic approaches and, ultimately, an innovative platform for the development of off-the-shelf CAR-T-cell products.

Noninvasive application of mesenchymal stem cell spheres derived from hESC accelerates wound healing in a CXCL12-CXCR4 axis-dependent manner.

Mesenchymal stem cells (MSC) derived from adult tissues effectively promote wound healing. However, MSC quality varies, and the quantity of MSC is limited, as MSC are acquired through donations. Moreover, the survival and functioning of dissociated MSC delivered to an inflammatory lesion are subject to challenges.Methods: Here, spheres (EMSCSp) generated from human embryonic stem cell-derived MSC (EMSC) were directly dropped onto excised wounds in mice; the effects of EMSCSp were compared to those of dissociated EMSC (EMSCDiss). Following transplantation, we measured the extent of wound closure, dissected the histological features of the wounds, determined transcriptomic changes in cells isolated from the treated and control wounds, and evaluated the molecular mechanism of the effects of EMSC.Results: The application of EMSCSp onto murine dermal wounds substantially increased survival and efficacy of EMSC compared to the topical application of EMSCDiss. RNA sequencing (RNA-Seq) of cells isolated from the wounds highlighted the involvement of CXCL12-CXCR4 signaling in the effects of EMSCSp, which was verified in EMSC via CXCL12 knockdown and in target cells (vascular endothelial cells, epithelial keratinocytes, and macrophages) via CXCR4 inhibition. Finally, we enhanced the biosafety of EMSCSp by engineering cells with an inducible suicide gene.Conclusions: Together, these data suggest the topical application of EMSCSp as an unlimited, quality-assured, safe, and noninvasive therapy for wound healing and the CXCL12-CXCR4 axis as a key player in this treatment.

T- and B-Cell Neogenesis Recovers Efficiently in Children with Acute Leukemia Given an Alpha-Beta T-Cell Depleted Haplo-HSCT Followed By Infusion of Donor T-Cells Genetically Modified with Inducible Caspase 9 Suicide Gene (BPX-501 cells)

Backgroundαβ T- and B-cell depleted HLA-haploidentical HSCT (αβ haplo-HSCT) is an alternative promptly available and virtually applicable to any children with malignant and non-malignant disorders lacking an HLA-matched donor or in need of an urgent allograft (Locatelli et al. 2017). Through this approach, recipients can benefit immediately after transplantation from the anti-leukemia effect mediated by donor natural killer (NK) and γδ T cells, which confers protection also against infections. Nonetheless, even with this type of HSCT, the recovery of adaptive immunity is suboptimal. We have developed a further refinement of the approach based on the post-transplant infusion of a titrated number of donor T cells transduced with the inducible suicide gene iC9 (BPX-501 cells). This strategy has the potential to accelerate the recovery of adaptive immunity and to restore an efficient T-cell mediated graft-versus-leukemia (GvL) effect without the risk of inducing uncontrollable graft-versus-host disease (GvHD). Since reconstitution of a naïve T- and B-cell repertoire plays a key role in the long-term ability to respond to a broad range of pathogens, as well as to tumor antigens, we quantified T-cell receptor and kappa chain-deleting recombination excision circles (TREC and KREC), which represent reliable surrogate of T and B-cell neogenesis respectively.Study design and patientsWe analyzed samples of 48 children (M/F: 26/22) with acute leukemia (31 ALL, 17 AML) given an ab T-cell and B-cell depleted haplo-HSCT after a myeloablative regimen followed by the infusion of a titrated number of donor BPX-501 cells (1 million/Kg) after a median of 27 days (range: 11-87). Median age at transplantation was 8.3 years (range, 0.9-18). Thirty-two and 16 patients did or did not receive a preparative regimen containing total body irradiation (TBI). Anti-T lymphocyte globulin (ATLG Grafalon®, Neovii Biotech, 12 mg/Kg) was administered from day -4 to -2 for preventing graft rejection and GvHD. Moreover, to reduce the risk of EBV-related PTLD, on day -1, patients received rituximab (200 mg/m2). We analyzed T-cell reconstitution by measuring the TREC, small DNA excision circles of the TCR d locus deleted during recombination of the a-locus and present in the majority of functional ab-T-cells (sjTREC), and during the TCR b-chain recombination (bTREC). We also analyzed B-cell reconstitution by measuring coding-joint (Cj), signal-joint KREC (sjKREC) which reflect newly produced naïve B cells and the mean number of B-cell division (n) using the formula n=LOG(Cj/sjKREC)/LOG2. We performed real-time quantitative PCR, as recently described (Arruda et al. 2018), on genomic DNA extracted from PBMC collected at 6 different time points (before and 1, 3, 6, 12 and 18 months after the allograft).ResultsRecovery of thymic function started at 3 months after transplant: sjTREC and bTREC amounts reached and overcame pre-transplant values at 6 months, continuing to increase until 18 months after HSCT. Number of B-cell divisions obtained by the measurement of cj and sjKREC found in peripheral blood matched with the newly-generated B-cells bone marrow output and showed the same kinetics of sj and bTREC, rising from the 3rd month after HSCT. All these data correlate with immunophenotyping findings, which show a progressive increase over time of both total CD3+ and ab-T-cells. Furthermore, we observed a negative impact on both T- and B-cell neogenesis played by aGvHD (occurring in 14 patients), which became significant from 3 months after HSCT for sjTREC (p=0.013) and at 6 months for sjKREC (p= 0.03). We did not observe any impact of the use of TBI during the preparative regimen on T- and B-cells generation in this cohort.ConclusionsBPX-501 cell infusion aims to contribute to the acceleration of immune reconstitution after a/b haplo-HSCT. In this study, we show that naïve T- and B- cells are quickly and efficiently generated in the early post-transplant period. This finding represents an interesting result considering the fully myeloablative conditioning regimens with the large use of TBI and the Ta/b and B-cells depletion (also in vivo by the administration of Rituximab) received by the patients. We observed an expected detrimental effect of aGvHD on immune reconstitution, but we emphasize the low frequency of this complication in this transplant setting despite the absence of any post-transplant pharmacological prophylaxis. DisclosuresFoster:Bellicum: Employment, Equity Ownership. Locatelli:bluebird bio: Consultancy; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Inducible Caspase9-mediated suicide gene for MSC-based cancer gene therapy

Cellular therapies based on mesenchymal stromal/stem cells (MSC) are promising strategies in regenerative medicine and oncology. Despite encouraging results, there is still some level of concerns on inoculating MSC in cancer patients. To face this issue, one possibility resides in engineering MSC by incorporating a suicide gene in order to control their fate once infused. Strategies based on Herpes Simplex Virus Thymidine Kinase (HSV-TK) and the Cytosine Deaminase genes have been developed and more recently a novel suicide gene, namely, iCasp9, has been proposed. This approach is based on a variant of human Caspase9 that binds with high affinity to a synthetic, bioinert small molecule (AP20187) leading to cell death. Based on this technology so far marginally applied to MSC, we tested the suitability of iCasp9 suicide strategy in MSC to further increase their safety. MSC have been transfected by a lentiviral vector carrying iCasp9 gene and then tested for viability after AP20187 treatment in comparison with mock-transfected cells. Moreover, accounting our anti-tumor approaches based on MSC expressing potent anti-cancer ligand TNF-Related Apoptosis-Inducing Ligand (TRAIL), we generated adipose MSC co-expressing iCasp9 and TRAIL successfully targeting an aggressive sarcoma type. These data show that anti-cancer and suicide mechanisms can coexist without affecting cells performance and hampering the tumoricidal activity mediated by TRAIL. In conclusion, this study originally indicates the suitability of combining a MSC-based anti-cancer gene approach with iCasp9 demonstrating efficiency and specificity.

Reporters to mark and eliminate basal or luminal epithelial cells in culture and in vivo.

The contribution of basal and luminal cells to cancer progression and metastasis is poorly understood. We report generation of reporter systems driven by either keratin-14 (K14) or keratin-8 (K8) promoter that not only express a fluorescent protein but also an inducible suicide gene. Transgenic mice express the reporter genes in the right cell compartments of mammary gland epithelia and respond to treatment with toxins. In addition, we engineered the reporters into 4T1 metastatic mouse tumor cell line and demonstrate that K14+ cells, but not K14− or K8+, are both highly invasive in three-dimensional (3D) culture and metastatic in vivo. Treatment of cells in culture, or tumors in mice, with reporter-targeting toxin inhibited both invasive behavior and metastasis in vivo. RNA sequencing (RNA-seq), secretome, and epigenome analysis of K14+ and K14− cells led to the identification of amphoterin-induced protein 2 (Amigo2) as a new cell invasion driver whose expression correlated with decreased relapse-free survival in patients with TP53 wild-type (WT) breast cancer.

Surfactant protein C dampens inflammation by decreasing JAK/STAT activation during lung repair.

Surfactant protein C (SPC), a key component of pulmonary surfactant, also plays a role in regulating inflammation. SPC deficiency in patients and mouse models is associated with increased inflammation and delayed repair, but the key drivers of SPC-regulated inflammation in response to injury are largely unknown. This study focuses on a new mechanism of SPC as an anti-inflammatory molecule using SPC-TK/SPC-KO (surfactant protein C-thymidine kinase/surfactant protein C knockout) mice, which represent a novel sterile injury model that mimics clinical acute respiratory distress syndrome (ARDS). SPC-TK mice express the inducible suicide gene thymidine kinase from by the SPC promoter, which targets alveolar type 2 (AT2) cells for depletion in response to ganciclovir (GCV). We compared GCV-induced injury and repair in SPC-TK mice that have normal endogenous SPC expression with SPC-TK/SPC-KO mice lacking SPC expression. In contrast to SPC-TK mice, SPC-TK/SPC-KO mice treated with GCV exhibited more severe inflammation, resulting in over 90% mortality; there was only 8% mortality of SPC-TK animals. SPC-TK/SPC-KO mice had highly elevated inflammatory cytokines and granulocyte infiltration in the bronchoalveolar lavage (BAL) fluid. Consistent with a proinflammatory phenotype, immunofluorescence revealed increased phosphorylated signal transduction and activation of transcription 3 (pSTAT3), suggesting enhanced Janus kinase (JAK)/STAT activation in inflammatory and AT2 cells of SPC-TK/SPC-KO mice. The level of suppressor of cytokine signaling 3, an anti-inflammatory mediator that decreases pSTAT3 signaling, was significantly decreased in the BAL fluid of SPC-TK/SPC-KO mice. Hyperactivation of pSTAT3 and inflammation were rescued by AZD1480, a JAK1/2 inhibitor. Our findings showing a novel role for SPC in regulating inflammation via JAK/STAT may have clinical applications.

Genetically engineered suicide gene in mesenchymal stem cells using a Tet-On system for anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) is the most aggressive malignancy of the thyroid, during which undifferentiated tumors arise from the thyroid follicular epithelium. ATC has a very poor prognosis due to its aggressive behavior and poor response to conventional therapies. Gene-directed enzyme/prodrug therapy using genetically engineered mesenchymal stromal cells (MSC) is a promising therapeutic strategy. The doxycycline (DOX)-controlled Tet inducible system is the most widely utilized regulatory system and could be a useful tool for therapeutic gene-based therapies. For example, use a synthetic “tetracycline-on” switch system to control the expression of the therapeutic gene thymidine kinase, which converts prodrugs to active drugs. The aim of this study was to develop therapeutic MSCs, harboring an inducible suicide gene, and to validate therapeutic gene expression using optical molecular imaging of ATC. We designed the Tet-On system using a retroviral vector expressing herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Mouse bone marrow-derived mesenchymal stromal cells (BM-MSC) were transduced using this system with (MSC-Tet-TK/Fluc2) or without (MSC-TK/Fluc) the Tet-On system. Transduced cells were screened and characterized. Engineered MSCs were co-cultured with ATC (CAL62/Rluc) cells in the presence of the prodrug ganciclovir (GCV) and stimulated with DOX. The efficiency of cell killing monitored by assessing Rluc (CAL62/Rluc) and Fluc (MSC-Tet-TK/Fluc and MSC-TK/Fluc) activities using IVIS imaging. Fluc activity increased in MSC-Tet-TK/Fluc cells in a dose dependent manner following DOX treatment (R2 = 0.95), whereas no signal was observed in untreated cells. eGFP could also be visualized after induction with DOX, and the HSV1-TK protein could be detected by western blotting. In MSC-TK/Fluc cells, the Fluc activity increased with increasing cell number (R2 = 0.98), and eGFP could be visualized by fluorescence microscopy. The Fluc activity and cell viability of MSC-Tet-TK/Fluc and MSC-TK/Fluc cells decreased significantly following GCV treatment. A bystander effect of the therapeutic cells confirmed in co-cultures of CAL62 cells, an anaplastic thyroid cancer cell line, with either MSC-Tet-TK/Fluc cells or MSC-TK/Fluc cells. The Rluc activity in MSC-Tet-TK/Fluc co-cultures, derived from the CAL62/Rluc cells, decreased significantly with GCV treatment of DOX treated cultures, whereas no significant changes were observed in untreated cultures. In addition, the Fluc activity of MSC-Tet-TK/Fluc cells also decreased significantly with DOX treatment whereas no signal was present in untreated cultures. A bystander effect also be demonstrated in co-cultures with MSC-TK/Fluc cells and CAL62/Rluc; both the Rluc activity and the Fluc activity were significantly decreased following GCV treatment. We have successfully developed a Tet-On system of gene-directed enzyme/prodrug delivery using MSCs. We confirmed the therapeutic bystander effect in CAL62/Rluc cells with respect to MSC-Tet-TK/Fluc and MSC-TK/Fluc cells after GCV treatment with and without DOX. Our results confirm the therapeutic efficiency of a suicide gene, with or without the Tet-On system, for ATC therapy. In addition, our findings provide an innovative therapeutic approach for using the Tet-On system to eradicate tumors by simple, repeated administration of MSC-Tet-TK/Fluc cells with DOX and GCV.

Generation of Genetically Engineered Precursor T-Cells From Human Umbilical Cord Blood Using an Optimized Alpharetroviral Vector Platform.

Retroviral engineering of hematopoietic stem cell-derived precursor T-cells (preTs) opens the possibility of targeted T-cell transfer across human leukocyte antigen (HLA)-barriers. Alpharetroviral vectors exhibit a more neutral integration pattern thereby reducing the risk of insertional mutagenesis. Cord blood-derived CD34+ cells were transduced and differentiated into preTs in vitro. Two promoters, elongation-factor-1-short-form, and a myeloproliferative sarcoma virus variant in combination with two commonly used envelopes were comparatively assessed choosing enhanced green fluorescent protein or a third-generation chimeric antigen receptor (CAR) against CD123 as gene of interest. Furthermore, the inducible suicide gene iCaspase 9 has been validated. Combining the sarcoma virus-derived promoter with a modified feline endogenous retrovirus envelope glycoprotein yielded in superior transgene expression and transduction rates. Fresh and previously frozen CD34+ cells showed similar transduction and expansion rates. Transgene-positive cells did neither show proliferative impairment nor alteration in their lymphoid differentiation profile. The sarcoma virus-derived promoter only could express sufficient levels of iCaspase 9 to mediate dimerizer-induced apoptosis. Finally, the CD123 CAR was efficiently expressed in CD34+ cells and proved to be functional when expressed on differentiated T-cells. Therefore, the transduction of CD34+ cells with alpharetroviral vectors represents a feasible and potentially safer approach for stem cell-based immunotherapies for cancer.

Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis.

Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

340. Development of a Nuclease Screen to Improve Cas9 Targeting Specificity

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) bacterial immunity targets invading nucleic acid sequences using an RNA guide (gRNA). The well-defined Cas9 from Streptococcus pyogenes’ type II CRISPR system has been utilized as a gene editing tool in a vast assortment of organisms. However, an inherent lack of specificity limits the potential of the current system and presents challenges to interpreting experimental results. A plasmid cleavage based screening platform has been developed with the intent to increase the fidelity of the Cas9 system. Utilizing both positive and negative selection, the screen can be used to identify variants of Cas9 or its gRNA with improved specificity. Positive selection of variants with activity towards the appropriate target site proceeds through the removal of a plasmid containing an inducible suicide gene. Conversely, negative selection of variants with reduced activity on off-target substrates involves the retention of a plasmid with an antibiotic resistance gene. Preliminary results suggest that a gRNA with an insertion in the scaffold region of the RNA can decrease the level of off-target cleavage. These results suggest that the screening platform has the potential to identify novel protein or RNA variants with greater targeting specificity compared to the naturally occurring components. A Cas9 system with enhanced fidelity will expand the potential applications of the technology and accelerate its ability to interrogate biological systems.

CD38 Chimeric Antigen Receptor Engineered T Cells As Therapeutic Tools for Multiple Myeloma

Chimeric Antigen Receptors (CARs) are engineered constructs consisting of an antibody-derived antigen recognition domain linked to intracellular T cell signaling domains. Cytotoxic T cells transduced to express tumor-reactive CARs are highly promising tools for immunotherapy of cancer. The CD38 molecule, with its high and homogenous expression on Multiple Myeloma (MM) tumor cells, is considered a suitable target for antibody therapy of MM. Prompted by this, we evaluated the feasibility and efficacy of targeting MM cells with CD38-CAR-transduced T cells (CD38-CAR T cells). To this end, we generated three different retroviral CAR constructs based on human CD38 antibodies as antigen recognition domain, CD3zeta and 41BB (CD137) as signaling domains and transduced them into PBMCs of a healthy donor. After in vitro selection and expansion, all CD38-CAR T cells, either unsorted or CD4/CD8 sorted, effectively lysed MM cell lines in a dose-, and CD38 expression-dependent manner, with a better efficacy for the CD8+ fraction. CD38-CAR T cells also effectively eradicated primary MM cells in the bone marrow mononuclear cells derived from MM patients, indicating their clinical relevancy. Although CD38-CAR T cells also displayed cytotoxic activity against the CD38+ fraction of mature monocytes and NK cells and to a lesser extent CD38+ B and T cells, they did not affect the outgrowth of CD34+ cells into various myeloid lineages. In addition,CD38-CAR T cell activity was effectively controllable by transducing them with a caspase 9-based inducible suicide gene. More interestingly, we discovered that the CD38-CAR T cells were themselves devoid of CD38 surface expression, indicating that CD38 was not essential for T cell expansion and function. Finally, in a novel in vivo xenotransplant model (UM9 cell line), in which myeloma cells were grown in a humanized bone marrow microenvironment, i.v. as well as intra tumor administration of CD38-CAR T cells established significant anti-tumor effects, proving that CD38-CAR endowed cytotoxic T lymphocytes, even with no CD38 expression, can efficiently migrate, infiltrate and eliminate human MM tumors growing in their natural niche. These results demonstrate the feasibility and potency of CAR mediated targeting of CD38+ MM cells. Optimization of CD38-CAR and suicide-gene control of CD38 CAR T cellsmay provide next steps towards safe clinical implementation of CD38-CAR T cell immune therapy. DisclosuresDrent:Genmab BV: Guest employee (unpaid) Other. Lammerts van Bueren:Genmab : Employment. Parren:Genmab: Employment, Equity Ownership. van de Donk:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding. Martens:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding. Lokhorst:Celgene: Research Funding; J&J: Research Funding; Genmab: Research Funding. Mutis:Celgene: Research Funding; J&J: Research Funding; Genmab BV: Research Funding.

Abstract 2796: Engineering multiple chimeric antigen receptors in T cells for the treatment of multiple myeloma

Abstract Multiple myeloma (MM) is an incurable plasma cell neoplasm accounting for 10% of all hematologic malignancies. With advanced treatments, the average survival has significantly improved. Recent progress in immunotherapy based on antibody and T cell receptor (TCR) engineering has shown great potential for treating cancer including MM. We hypothesize that immune cell engineering that overcomes immune suppression in the BM may completely eradicate residual cancer cells. To test our hypothesis, we have developed a streamlined approach to generate lentiviral constructs with chimeric antigen receptors (CAR) of T cells based on single chain antibodies that target multiple MM antigens including CD138, CD269 (mature B cell marker BCMA, or TNFRSF17), CD317 (hm1.24), and CD319 (CS1). To increase safety, we also incorporated a self-destructive, inducible suicide caspase 9 gene, which is fused with the CAR. Memory CD8 T cells were isolated from BM specimens of MM patients and immortalized for prolonged in vitro studies. These primary BM T cells were transduced using lentiviral vectors encoding the MM-targeting CARs, and co-cultured with MM cell lines to measure killing effect. The MM-specific killing activity was demonstrated based on annexin V and propidium iodide staining and flow cytometry analysis for quantitation of cell death. In transient (24 hours) killing assays, both CD138 and CD 319 CARs displayed moderate MM killing activities, at 48% and 36%, respectively, when using a CAR T effector to MM Target (E/T) ratio of 0.5/1. Interestingly, a synergistic MM killing effect (a five fold increase at an E/T ratio of 1/1) was observed when these two CARs were used in combination. On the other hand, CD269 and CD317 CAR T cells displayed marked MM killing activities, approximately 80% for both at an E/T ratio of 0.5/1 in 24 hours. In a long term CAR T/MM co-culture setting at E/T ratios of 0.5/1 and 1/1, we observed near complete MM killing (100%) by all four MM-targeting CAR T cells in two weeks. The self-destructive function of the inducible suicide gene in the CAR T cells was also demonstrated in the long-term co-cultures. Interestingly, upon killing of the CAR T cells by apoptotic induction, residual MM cells rebounded from the CD319 CAR T/MM co-culture, but not the other three CAR T/MM co-cultures, suggesting that rapid and efficient killing of MM cells by CAR T cells is necessary for complete eradication of MM cells. In summary, all four CARs demonstrated MM killing activities in short term and long term T cell co-cultures. Combination of multiple CARs may be necessary for effective suppression of MM residual disease in vivo and to prevent antigenic escape phenomenon. Together with the inducible apoptotic gene design, increased safety and efficiency for the CAR-based immunotherapy may be attained in future clinical trials. Citation Format: Lung-Ji Chang, Yuchen Liu, Jan S. Moreb. Engineering multiple chimeric antigen receptors in T cells for the treatment of multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2796. doi:10.1158/1538-7445.AM2014-2796

<em>piggyBac</em> Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect, mammalian, and human cells6 including primary human T cells. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency. Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized. Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry. PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb). Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

The Effects of B16-F10 Melanoma Tumors on Iron Biomarkers and Anemia of Inflammation In Vivo,

Abstract 3165Anemia of inflammation (AI) is associated with the immune response that occurs when inflammatory diseases interfere with the body’s ability to absorb and use iron. Cancer and other chronic illnesses produce inflammatory cytokines, some of which can diminish the production of red blood cells by affecting erythropoietin levels. Cancer cells might also modify system iron homeostasis for their survival and proliferation. To date only a few studies have addressed the role of cancer in AI. For this reason we selected the tumor cell line B16−F10, which expresses the transferrin receptor−1 (TfR1), and whose growth can be modulated by iron−chelating agents. Cancer cell line B16−F10 also triggers a profound anemia as the tumor progresses. However, the rapid expansion of the tumor poorly represent human models and limits the analyses that can be done to evaluate the role of iron metabolism and inflammation in the etiology of the anemia. For this reason we modified the tumor cell line B16−F10 melanoma with an inducible suicide gene to prevent tumors from expanding too quickly. Using this model in comparison with controls, effects on inflammation and iron metabolism were assessed in tumorigenic mice at different time points.B16−F10 cells were modified with an oncoretroviral vector that encodes an engineered fusion molecule that activates a proapoptotic molecule when used with a homodimerizer molecule (AP). B16−F10 cells with an inducible suicide gene were injected into C57BL/6 mice. After the appropriate tumor size was obtained the mice were injected with the inducer to control the growth of the tumor. Bone marrow and spleen cells from mice were analyzed by flow cytometry (FACS) to assess erythropoiesis. Serum was analyzed for iron content and transferrin saturation by spectroscopy and for markers of inflammation by cytokine microarray. Several organs were also collected for analysis of inflammation and iron− related gene expression.Mice analyzed 20 to 28 days after injection of B16−F10 cells and without treatment exhibited large tumors, had lower hemoglobin (7.1±1.5 vs. 12.9±1.6 g/dL) and higher reticulocytes (15.28±3.9 vs. 2.13±0.26 × 105 cells/mL) compared to control mice (p<0.05). Analysis of inflammatory cytokines and FACS carried out using CD44, Ter119 and CD71 markers suggested that anemia was associated with inflammation and ineffective erythropoiesis both in the spleen and bone marrow (BM). Serum iron and Tf saturation levels were not significantly altered. Mice treated with 5 mg/kg of AP eliminated or shrunk the tumors to undetectable sizes and had similar hematological parameters as control mice. Tumorigenic mice treated with less than 3 mg/kg of AP reduced tumor growth but did not eradicate the tumor. These animals still exhibited anemia, abnormal erythropoiesis but had lower reticulocyte counts than untreated mice with similar tumor sizes analyzed at earlier time points.As hemoglobin decreased in mice, apoptosis and ROS increased in erythroid populations in the BM and spleen. Inspection of BM and spleen indicated absence of metastasis. Altogether, these analyses suggested that tumor formation was indirectly responsible for the cell death of the erythroid cells, most likely by an inflammatory mechanism. Compared to normal mice, RT-qPCR analysis indicated that hepcidin, ferroportin, divalent metal transporter-1, and TfR1 were reduced in the liver of mice at day 14 and even more at day 28 during tumor development. In contrast, inflammatory markers including interleukin-6 (IL6) and IL6R were increased significantly in the serum or liver respectively, during the early phases of tumor development, but not at day 28 post-tumor injection. Altogether, these analyses suggest that an inflammatory mechanism affects erythropoiesis, but that the anemia has a dominant effect on iron metabolism, repressing hepcidin expression.In conclusion, the ineffective erythropoiesis is likely responsible for the anemia observed during tumor progression mediated by B16−F10 melanoma cells. Both apoptosis and ROS of progenitor erythroid cells appear to contribute to the development of anemia, with IL6 and other inflammatory markers as potential players in this process. This study also successfully showed tumor growth can be controlled with an inducible suicide gene, which is useful for analyzing inflammation and iron metabolism effects in mice with more slowly progressing melanoma tumors. Disclosures:No relevant conflicts of interest to declare.

Genetic Modification of Multi Leukemia Antigen-Specific Cytotoxic T Lymphocytes (CTL) to Enhance In Vivo Safety and Persistency

Abstract 644Adoptive transfer of ex-vivo expanded leukemia-specific T cells has been clinically evaluated for its ability to produce anti tumor effects without causing graft-versus-host disease (GVHD). Previous protocols, however, allowed the expansion only of HLA class I-restricted CD8 T cells, which were short-lived and permitted tumor immune escape since they recognized only a single epitope/antigen target.To minimize tumor immune escape and extend this therapeutic alternative to more patients irrespective of HLA type we have developed a novel strategy to produce single cultures of in vitro generated T cell lines containing tumor-cytotoxic T cells from both CD4+ and CD8+ populations with specificity for a multiplicity of epitopes on several tumor associated antigens (TAAs) frequently expressed by myeloid leukemias.Tumor-directed T cells were activated in vitro using dendritic cells (DCs) loaded with peptide libraries (pepmixes) spanning Proteinase 3 (Pr3), PRAME, and WT1 and expanded in the presence of a cocktail of Th1-polarizing, pro-survival and pro-proliferative cytokines (IL7, 12, 15 and 27). This approach consistently generated TAA-specific cytotoxic T lymphocytes (CTLs) with simultaneous reactivity against PRAME (mean 651+/−69 spot forming units (SFU)/1×106 CTLs), WT1 (mean 156+/−20 SFU/1×106 CTLs) and PR3 (mean 52.3+/−8.8 SFU/1×106 CTLs) in IFNg ELIspot assays (n=10). These lines were polyclonal, comprising antigen-specific CD4+ (mean 32.5+/−2.3%) and CD8+ (mean 38.3+/−4%) T cells. Functionally, these CTLs killed autologous PHA blasts pulsed with PRAME (mean specific lysis 67+/−5% SEM), WT-1 (mean 54+/− 5%) and PR3 pepmixes (mean 16+/−6%, n=6) (E:T of 40:1). In addition, TAA CTLs killed the partially (1-3 alleles) HLA class I or HLA class II matched whole antigen-expressing AML cell line THP1 in coculture assays (n=5).The in vivo anti-tumor activity of multiTAA-CTLs was tested using a SCID mouse tumor model. After subcutaneous engraftment of FFLuc transduced THP-1 cells, mice were treated with either 1×107 multiTAA-CTLs or control EBV-CTLs generated from the same partially HLA-matched donor (sharing HLA-A2, DRB1-01,DQB1-05) plus 1000U of IL2 (5 times per week). Tumor signals as measured by bioluminescence using an in vivo imaging system rapidly increased in untreated and EBV-CTL treated groups, whereas mice treated with TAA specific CTLs were able to control tumor growth (p<0.05) and significantly improve overall survival (log rank p=0.008).Next, we transduced multiTAA-CTL with a retroviral vector (SFG-hIL-7R/iCasp9) encoding IL7Ralpha and the inducible suicide gene iCaspase9. This genetic modification improved the in vivo persistence, anti-tumor potency of multiTAA-CTL by restoring their response to homeostatic IL7. Furthermore we improve safety with the potential to control unwanted proliferation of the transgenic cells and to treat potentially occurring autoimmune disease caused by targeting self antigens through the administration of a small molecule chemical inducer of dimerization (CID) (AP20187) which activates the suicide gene.Transduction of multiTAA-CTLs did not adversely affect the antigen specificity of the product as evaluated by IFNg ELIspot (528.5 SFU/1×106 CTL transduced vs. 573.9 SFU/1×106 CTL non transduced, PRAME, 86.7 vs.46.5 SFU/1×106 CTL, Pr3 and 129.2 vs 130.2 SFU/1×106 CTL and WT1 transduced vs. non transduced); and cytolytic activity against peptide-pulsed PHA blasts as well as whole antigen presenting tumor cell lines was maintained. In the presence of IL-7, transgenic multiTAA-CTLs expanded significantly (transduced cells increased from 55%+/1% to 89%+/−1%). Such expansion was not observed when these cells were cultured in the presence of IL-15 (decrease 55%+/1% to 34.6%+/−3.5%) (n=8). Proliferation assays confirmed that only IL7/casp9 engineered multiTAA-CTLs were able to proliferate in the presence of IL-7 (84638 CPM vs. 14923 CPM transduced vs non-transduced). Upon activation of iCaspase9 by of CID (AP20187), the proliferation of multiTAA-CTLs was halted and a decrease of transgenic cells from 89% to 3% with was detected within 24 hrs (n=7).Hence CTLs can be prepared that simultaneously target multiple TAA antigens expressed by myeloid malignancies, reducing the risk of tumor immune escape. In addition these tumor CTLs can be genetically modified to safely improve their in vivo survival, and persistence. Disclosures:No relevant conflicts of interest to declare.

Manipulating Tumor Associated Macrophages (TAM) in a Mouse Model of B-Cell Non-Hodgkin Lymphoma (NHL)

Abstract 1657 Introduction:In vitro stimulation of macrophages can produce classically activated (M1) or alternatively activated (M2) phenotypes. In various solid tumors, tumor associated macrophages (TAM) have been described as approximating an M2 phenotype and are potentially pro-tumoral. We are testing the hypothesis that TAM play an important role in the progression of NHL, and might therefore constitute a rational and effective therapeutic target.Our aim is to investigate tumor-macrophage interactions by manipulating macrophage numbers and phenotype in a transplantable mouse lymphoma. Experimental approach:We used as a model system B-cell lymphoma arising in Em-myc/bcl-2 transgenic mice, which, when intravenously injected into C57BL/6 mice produces disseminated lymphoma.Lymphoma growth was assessed by measuring lymph node weight, and cross-sectional tumor area in tissue sections, as well as by flow cytometry. Gene expression changes were determined by real-time PCR in whole lymph nodes or sorted macrophages in control mice or mice with lymphoma, and following interventions to manipulate macrophage populations.Changes to the cellular composition of the immune microenvironment were assessed by flow cytometry of lymph nodes and blood, and by immunohistochemistry. Methods and results:The consequences of widespread monocyte and macrophage depletion were first investigated using intravenous injection of the liposome-encapsulated bisphosphonate, sodium clodronate (Liposomal Clodronate, LC).Intravenous LC successfully depleted macrophages in the bone marrow, lymph nodes, liver and spleen, and correlated with significantly reduced lymphoma mass by weight and flow cytometry, compared to controls (p=0.0058).Next we examined MaFIA mice (Macrophage Fas Induced Apoptosis, Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) that combine the expression of an enhanced green fluorescent protein on cells with the macrophage-specific csf-1 receptor, with a drug (dimerizer, AP20187) inducible suicide gene leading to Fas-mediated apoptosis in resting and cycling cells of the macrophage lineage.We observed the effects of this non-pharmacological method of macrophage depletion on lymphoma progression.Lymph node macrophage numbers are not reduced in normal healthy mice treated with the dimerizer, but were significantly reduced in the infiltrated lymph nodes of mice with lymphoma given the dimerizer. This was associated with a reduced lymphoma mass (p=0.044).Subsequent studies employed adoptive transfer of syngeneic bone marrow derived macrophages (BMDM), in vitro polarized to M1, M2 and model-specific TAM phenotypes; the purpose being to test whether supplementing the TAM population might invigorate lymphoma growth, and whether administering M1 phenotype macrophages might lead to anti-tumor effects.Adoptive transfer of M1-polarized BMDM attenuated lymphoma growth, and transfer of TAM augmented lymphoma growth (p=0.0003).Having gathered evidence that reducing TAM numbers might slow lymphoma growth, we explored a novel approach to targeting this macrophage sub-population in lymphoma, with translational potential for human studies. More specific targeting of TAM, and relative sparing of physiological monocytes and resident tissue macrophages, was attempted by targeting the CSF1 receptor with an orally available highly selective small molecule inhibitor. Current literature suggests this might reduce both monocyte trafficking to areas of malignancy, and maturation of monocytes to TAM. This strategy was used against growing lymphomas, resulting in an attenuated TAM population (p=0.0009) and reduced lymphoma mass (p=0.0036), whilst not depleting the circulating monocyte pool.In vitro cytotoxicity studies confirmed that neither LC, nor the CSF1R inhibitor nor AP20187 dimerizer caused significant direct lymphoma cell death, confirming that the observed in vivo attenuation of lymphoma growth likely resulted from indirect phenomena, such as TAM depletion. Conclusion:By evaluating a variety of pharmacological, genetic and cellular therapy strategies we have generated a strong body of evidence supporting a causal relationship between TAM abundance and lymphoma progression in this model. These data support the hypothesis that targeting TAM may well provide an attractive therapeutic opportunity to improve outcome in human B-cell lymphomas. Disclosures:Hagemann:Astra Zeneca and the Medical Research Council: In receipt of a joint studentship with Astra Zeneca and the UK Medical Research Council. Gribben:Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.