mRNA Electroporation Research Articles

MRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5.

Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV (‘Berlin patient’). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method.

562. Modification of TCR Specificity by TALEN and CRISPR

Adoptive T-cell transfer of T-cells with transgenic high avidity T-cell receptors (TCR) holds a lot of promise as therapeutic approach, however comprising certain challenges. Endogenous and transferred TCR-chains compete for surface expression and may pair inappropriately, potentially leading to autoimmunity. Designer nucleases like transcription activator-like effector nucleases (TALEN) and RNA-guided nucleases of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system introduce specific DNA double strand breaks (DSB) at their target sites. This ability can be exploited for targeted genome editing. DSB repair through non-homologous end joining (NHEJ) can result in permanent gene knockout. Alternatively, if a homology-containing donor template is provided, transgenes can be inserted into the specific target locus by homology directed repair (HDR). In order to disrupt endogenous TCR expression, we assembled seven TALEN monomers and two guide RNAs targeting the constant regions of the TCR α-chain (TRAC) and the TCR β-chain (TRBC1 and TRBC2), respectively. Here we show the specific DSB induction by TALEN and CRISPR-Cas nucleases at their target sites, examined in K562 and T-cells using Cel1-assay and deep sequencing. Compared to nucleofection of TALEN-expressing plasmids, electroporation of mRNA considerably improved TCR knockout efficiency in primary T-cells. Using TRAC-TALEN and TRBC-TALEN mRNA, surface CD3 expression was successfully eliminated in about 75% and 60% of T-cells, respectively. The nuclease specificity was examined by identifying integrase-deficient lentiviral vector (IDLV)-marked DSB using LAM-PCR and deep sequencing. Although clustered IDLV integration sites were detected at the TALEN and CRISPR target sites, we did not detect any off-target activity, indicating high specificity. Additionally, K562 cells were nucleofected with TALEN-expressing plasmids and a donor template containing a GFP expression cassette flanked by 800bp homologous sequences to the TRAC locus. Thereby we were able to verify targeted gene addition through HDR of TALEN-mediated DSB in about 10% of treated cells. TALEN and CRISPR that show high efficiency and specificity to their target sequence will be used for generating T-cells with high avidity TCR.

565. CCR5-Uco-TALEN – A Novel Transcription Activator-Like Effector Nuclease That Mediates High-Efficiency Knockout of HIV Co-Receptor CCR5 in Primary T Cells After mRNA Transfection

Besides its physiological role, the chemokine receptor CCR5 plays an essential part during HIV infection, acting as the co-receptor for so-called R5-tropic strains that usually mediate initial infection. Notably, homozygosity for CCR5D32, a natural deletion variant of CCR5, largely confers resistance towards HIV infection. Consequently, CCR5 has been proposed as a promising target for HIV therapy. The interest in using CCR5 as a therapeutic target has increased even further after the first report on the cure from HIV in an individual (“Berlin patient”) who received an allogeneic stem cell graft from a CCR5D32-homozygous donor to treat his acute leukemia. Since allogeneic transplantation is not a realistic treatment scenario for HIV, genetic disruption of CCR5 by the means of designer nucleases was proposed as an auspicious gene-therapy approach. We developed a novel transcription activator-like effector nuclease (TALEN), CCR5-Uco-TALEN for highly specific CCR5 knockout. We established efficient TALEN delivery into primary T lymphocytes using mRNA electroporation, a gentle and truly transient gene-transfer technique. After mRNA transfection, CCR5-Uco-TALEN routinely mediated high-rate CCR5 knockout (>90% in PM1 and >50 % in primary T cells) combined with low off-target activity, as demonstrated by flow cytometry, next-generation sequencing and a newly devised, very handy digital-PCR technique. Importantly, CCR5-edited cells were not only resistant towards HIV-derived lentiviral vectors, but also protected from infection with the wild-type CCR5-tropic HIV-1BaL strain. Indeed, 12-day exposure to HIV-1BaL resulted in almost complete suppression of viral replication as well as selection of CCR5-gene edited T cells. In summary, we have introduced a novel TALEN for high-efficiency targeted CCR5 knockout. The proposed mRNA-based gene-editing protocol does not rely on gene-modified organisms and is thus highly compatible with large-scale production and GMP requirements. Together, CCR5-Uco-TALEN and the proposed mRNA-based transfection strategy should therefore be excellently suited for clinical translation.

285. Exploring On-Target Hematopoietic Toxicity With Highly Potent, High Affinity FRβ-Specific CAR T Cells for AML

On-target, off-tumor recognition of antigen in normal tissues is one of the largest safety concerns in chimeric antigen receptor (CAR) T cell therapy. Careful assessment of target protein expression by flow cytometry or IHC can provide helpful data for prediction of potential toxicity. However, high affinity reagents should be carefully chosen in order to assure the most sensitive and thorough investigation of target antigen levels, which may be low in normal tissues. We are currently optimizing our folate receptor beta (FRβ)-directed CAR T cell platform for acute myeloid leukemia (AML) and have isolated a high affinity antibody single-chain-variable fragment (scFv) for production of CAR T cells. The high affinity (m923, 2.48nM KD) CAR T cells exhibited greatly enhanced reactivity against FRβ(+) AML in vitro and in vivo compared to previously generated lower affinity (m909, 54.3nM KD) FRβ-specific CAR T cells. However, higher affinity reagents pose the potential for increased risk of toxicity directed against normal tissues. To most accurately predict m923 CAR T cell toxicity, we utilized high affinity m923 IgG for analysis of FRβ expression in normal hematopoietic tissues by flow cytometry. We were able to detect high levels of FRβ in peripheral blood monocytes as well as low levels in myeloid-lineage bone marrow progenitor cells which were non-detectable using m909 IgG. Notably, CD34(+) bone marrow hematopoietic stem cells (HSCs) displayed very low expression of FRβ and m923 CAR T cells did not inhibit HSC colony formation in classic CFU assays. We next examined the ability of m923 CAR T cells to target whole bone marrow populations. Upon co-culture, m923 CAR T cells were able to eliminate FRβ(+) myeloid progenitors while sparing neighboring FRβ(-) bone marrow cells. We hypothesized that, because high affinity FRβ-specific CAR T cells did not impact bone marrow HSCs and non-myeloid-committed progenitors, short-term treatment with CAR T cells may allow for tumor clearance, following which any affected myeloid populations could be reconstituted from healthy HSCs. Therefore, we investigated transient expression via mRNA electroporation of the m923 CAR platform. mRNA-CAR T cells retained effective anti-tumor activity. Our results highlight the importance of antibody affinity for assessment of target protein during CAR development. In addition, we report a highly potent, high affinity FRβ-specific CAR T cell platform that does not target HSCs, and, when delivered transiently through mRNA electroporation, could allow for AML tumor destruction while providing a decreased risk for long-term myeloid toxicity.

298. Transfection of Neutrophils from X-Linked Chronic Granulomatous Disease Patients with gp91phox mRNA Restores Oxidase Activity with High Efficiency and Viability

Chronic granulomatous disease (CGD) is an inherited immune deficiency due to defective phagocyte NADPH oxidase required for production of antimicrobial oxidants. Fungal or bacterial infections in CGD may persist despite intensive and prolonged antibiotic therapy. In settings where conventional antibiotic therapy and/or surgical debridement do not control infection, use of allogeneic unmatched donor granulocyte transfusions has been therapeutic. However, CGD patients treated with granulocyte transfusions may develop high titers of anti-HLA antibodies, restricting use of allogeneic granulocyte transfusions and further complicating potentially curative bone marrow transplant. Using the CGD patient as an autologous donor for granulocyte transfusions would be a helpful solution.Here we demonstrate highly efficient functional correction of the functional defect in CGD by transfection of patient's own granulocytes with mRNA while retaining viability using a scalable, cGMP-compliant MaxCyte® GT™ electroporation system capable of handling clinically relevant numbers of cells (>1010cells).Freshly collected granulocytes from normal volunteers were electroporated using the MaxCyte electroporation system with eGFP-mRNA on the day of collection. Flow cytometry of demonstrated that >90% of neutrophils expressed eGFP with viability exceeding 90% at 24 hrs after electroporation. Phagocyte NADPH oxidase activity of the normal neutrophils was retained as well as bactericidal activity against Burkolderia cepacia, a CGD bacterial pathogen. Antibacterial activity of electroporated granulocytes was comparable to unmanipulated granulocytes confirming retention of cellular function following the electroporation. Finally, electroporated eGFP positive human neutrophils injected into CGD mice were shown to remain in circulation as well as migrate to the peritoneal cavity following thioglycollate IP injection and there were no adverse effects observed in the mice.Electroporation of gp91phox mRNA into human X-linked CGD (gp91phox-deficient) patient neutrophils resulted in expression of gp91phox protein in >70% of cells with >90% viability at 24 hours after treatment. Using the dihydrorhodamine assay of phagocyte NADPH oxidase activity, >70% of neutrophils remained functionally corrected 2 days after transfection. Other studies of cellular function in vitro and in vivo with electroporated corrected CGD neutrophils are in progress. In summary, we show the feasibility of restoring the function of CGD patient-autologous neutrophils via electroporation of mRNA encoding the defective gene product. This approach may have potential clinical utility in the management of severe chronic bacterial and fungal infections in CGD but also short-term correction of other hematopoietic gene diseases.

239. Preclinical Studies for the First Hematopoietic Stem Cell (HSC) Gene Editing Trial: Phase 1 Study of Beta-Thalassemia With Autologous Transplantation of Zinc Finger Nuclease-Treated HSC To Upregulate Fetal Hemoglobin

Beta-thalassemia (β-thal) and sickle cell disease (SCD) are monogenic diseases caused by mutations in the adult β-globin gene. Allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment, but its application is limited by: (i) a paucity of HLA-matched donors and (ii) a fixed risk of graft vs host disease (GvHD) and transplant-related mortality. Sustained elevation of fetal hemoglobin (HbF) has a very strong clinical effect in both thalassemia and SCD with curative potential. HbF levels are under genetic control, and alleles of the transcription factor BCL11A that lower its expression result in elevation of HbF. BCL11A is thus an attractive target for gene editing of autologous hematopoietic stem cell (HSC) for HCT in β-hemoglobinopathies.We have designed zinc finger nucleases (ZFNs) that when transiently introduced into stem cells ex vivo can efficiently disrupt the BCL11A gene in hematopoietic stem cells (HSCs) by introduction of mutations following target site cleavage and non-homologous end joining. We have developed a clinical scale GMP process for mRNA electroporation of BC11A ZFNs into HSCs, and show that erythrocytes generated ex vivo from BCL11A-modified HSC have elevated fetal globin mRNA, and over 40% of all Hb is represented by HbF. A similar increase in HbF was seen in modified HSCs from subjects with β-thalassemia. Engraftment and differentiation potential of BCL11a modified and control HSCs in NSG mice were comparable. Furthermore, following engraftment into NSG mice, HSCs placed into erythropoiesis cultures maintained increased HbF levels. Preclinical safety studies included soft agar transformation, karyotyping, and double-strand break quantification following ZFN modification. Both bioinformatics guided and a novel, unbiased, oligonucleotide capture method were used to confirm low levels of off-target activity in BCL11A modified HSC as gauged by deep sequencing. In vivo carcinogenicity was evaluated by engraftment of HSC modified by BCL11A ZFNs in NSG mice. Engraftment and multilineage differentiation were comparable between groups and no evidence for carcinogenicity was found.The primary objective of the Phase I clinical study is to evaluate the safety of autologous BCL11A gene edited HSPC infusion after myeloablative (full dose busulfan) conditioning in subjects with transfusion dependent β-thalassemia major. The secondary objectives are to assess hematologic and immunological reconstitution, and to measure the treatment effects on anemia, RBC transfusion frequency, and iron burden.

Engineering monocyte-derived dendritic cells to secrete interferon-α enhances their ability to promote adaptive and innate anti-tumor immune effector functions.

Dendritic cell (DC) vaccination has demonstrated potential in clinical trials as a new effective cancer treatment, but objective and durable clinical responses are confined to a minority of patients. Interferon (IFN)-α, a type-I IFN, can bolster anti-tumor immunity by restoring or increasing the function of DCs, T cells and natural killer (NK) cells. Moreover, type-I IFN signaling on DCs was found to be essential in mice for tumor rejection by the innate and adaptive immune system. Targeted delivery of IFN-α by DCs to immune cells could boost the generation of anti-tumor immunity, while avoiding the side effects frequently associated with systemic administration. Naturally circulating plasmacytoid DCs, major producers of type-I IFN, were already shown capable of inducing tumor antigen-specific T cell responses in cancer patients without severe toxicity, but their limited number complicates their use in cancer vaccination. In the present work, we hypothesized that engineering easily generated human monocyte-derived mature DCs to secrete IFN-α using mRNA electroporation enhances their ability to promote adaptive and innate anti-tumor immunity. Our results show that IFN-α mRNA electroporation of DCs significantly increases the stimulation of tumor antigen-specific cytotoxic T cell as well as anti-tumor NK cell effector functions in vitro through high levels of IFN-α secretion. Altogether, our findings mark IFN-α mRNA-electroporated DCs as potent inducers of both adaptive and innate anti-tumor immunity and pave the way for clinical trial evaluation in cancer patients.

Inclusion of an IgG1-Fc spacer abrogates efficacy of CD19 CAR T cells in a xenograft mouse model.

Cancer therapy with T cells expressing chimeric antigen receptors (CARs) has produced remarkable clinical responses in recent trials, but also severe side effects. Whereas most protocols use permanently reprogrammed T cells, we have developed a platform for transient CAR expression by mRNA electroporation. This approach may be useful for safe clinical testing of novel receptors, or when a temporary treatment period is desirable. Herein, we investigated therapy with transiently redirected T cells in vitro and in a xenograft mouse model. We constructed a series of CD19-specific CARs with different spacers and co-stimulatory domains (CD28, OX40 or CD28-OX40). The CAR constructs all conferred T cells with potent CD19-specific activity in vitro. Unexpectedly, the constructs incorporating a commonly used IgG1-CH2CH3 spacer showed lack of anti-leukemia activity in vivo and induced severe, partly CD19-independent toxicity. By contrast, identical CAR constructs without the CH2-domain eradicated leukemia in vivo, without notable toxicity. Follow-up studies demonstrated that the CH2CH3-spacer bound soluble mouse Fcγ-receptor I and mediated off-target T-cell activation towards murine macrophages. Our findings highlight the importance of non-signalling CAR elements and of in vivo studies. Finally, the results show that transiently redirected T cells control leukemia in mice and support the rationale for developing an mRNA-CAR platform.

Electroporated Antigen-Encoding mRNA Is Not a Danger Signal to Human Mature Monocyte-Derived Dendritic Cells.

For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs) is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs). However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs' immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1), and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs.

Clinical-Grade mRNA Electroporation of NK Cells: A Novel and Highly Efficient Method to Genetically Reprogram Human NK Cells for Cancer Immunotherapy

Natural killer (NK) cells are immune cells involved in the defense against cancer. They have also been shown to induce strong anti-tumor responses in the setting of hematopoietic stem cell transplantation and in early clinical trials on adoptive NK cell transfer. Several methods to expand large numbers of clinical-grade NK cells have been developed for trials exploring adoptive NK cell immunotherapy for cancer. However, long-term culturing of NK cells often lead to undesirable phenotypic changes that may compromise their homing capacity and cytotoxic function, and can also lead to senescence compromising in vivo longevity. Introducing genes into NK cells that improve their in vivo viability, cytotoxicity, and ability to home to disease sites could improve the efficacy of NK cell-based immunotherapy. Previously, we and others have shown that genetic manipulation of NK cells through viral transduction is challenging, typically resulting in substantial reduction in NK cell viability and low transduction efficiency. mRNA transfection is an alternative strategy to genetically modify ex vivo expanded NK cells that may overcome limitations of viral transduction. Here we present data characterizing the transgene expression, viability, proliferative capacity, phenotype and cytotoxic function of clinical-grade ex vivo expanded human NK cells following mRNA electroporation using the GMP compliant MaxCyte system. Using unmodified mRNA coding for GFP and the cell surface marker CD34, we established that this technology resulted in rapid and highly efficient protein expression in NK cells without compromising their viability and cytotoxic function (Figure). NK cells electroporated with GFP mRNA rapidly became GFP positive and remained fluorescent for more than two weeks. Following transfection of CD34 mRNA, nearly 100% of NK cells expressed CD34 that remained detectable on the cell surface for up to five days, without affecting viability amongst transfected cells. With the exception of a slight reduction in proliferative capacity compared to controls, no negative impacts of mRNA electroporation using the MaxCyte platform were observed. Transfection of expanded NK cells did not alter expression of twenty cellular markers as assessed by flow cytometry, including activating and inhibitory NK cell receptors and death receptor ligands such as TRAIL. Further, electroporated NK cells maintained high cytotoxic function against K562 cells and multiple myeloma cells (Figure). In conclusion, mRNA electroporation of ex vivo expanded NK cells using the clinical-grade MaxCyte transfection system is highly efficient and opens numerous new possibilities to advancethe field of NK cell-based cancer immunotherapy. [Display omitted] DisclosuresLi:MaxCyte Inc.: Employment, Patents & Royalties. Peshwa:MaxCyte Inc.: Employment, Patents & Royalties; Indian Biomedical Association: Membership on an entity’s Board of Directors or advisory committees; Epidarex Capital: Membership on an entity’s Board of Directors or advisory committees; BioMetrx LLC: Membership on an entity’s Board of Directors or advisory committees.

Triggering of NF-κB in cytokine-matured human DCs generates superior DCs for T-cell priming in cancer immunotherapy.

Understanding the signaling that governs the immunogenicity of human dendritic cells (DCs) is a prerequisite for improving DC-based therapeutic vaccination strategies, in which the ability of DCs to induce robust and lasting Ag-specific CTL responses is of critical importance. Cytokine-matured DCs are regularly used, but to induce memory-type CTLs, they require additional activation stimuli, such as CD4+ T-cell help or TLR activation. One common denominator of these stimuli is the activation of NF-κB. Here, we show that human monocyte-derived, cytokine cocktail-matured DCs transfected with constitutively active mutants of IκB kinases (caIKKs) by mRNA electroporation, further upregulated maturation markers, and secreted enhanced amounts of cytokines, including IL-12p70, which was produced for more than 48 h after transfection. Most importantly, cytotoxic T cells induced by caIKK-transfected DCs combined high CD27 expression, indicating a more memory-like phenotype, and a markedly enhanced secondary expandability with a high lytic capacity. In contrast, CTLs primed and expanded with unmodified cytokine cocktail-matured DCs did not maintain their proliferative capacity upon repetitive stimulations. We hypothesize that "designer" DCs expressing constitutively active IκB kinases will prove highly immunogenic also in vivo and possibly emerge as a new strategy to improve the clinical efficacy of therapeutic vaccinations against cancer and other chronic diseases.

Genetic Modification of T Cells

Gene transfer technology has advanced rapidly from simple physical-chemical laboratory methods in the 1970s and 1980s to the sophisticated viral and nonviral methods currently in clinical practice. Herein, we review 4 gene transfer methodologies applied in human gene therapy clinical trials transferring chimeric antigen receptors into T cells for the treatment of B-cell malignancies. The 4 methods include 2 viral vector gene transfer technologies, gamma retroviral vectors and lentiviral vectors, and 2 nonviral methods, transposons and mRNA electroporation.

Interference with PD-L1/PD-1 co-stimulation during antigen presentation enhances the multifunctionality of antigen-specific T cells

The release of cytokines by T cells strongly defines their functional activity in vivo. The ability to produce multiple cytokines has been associated with beneficial immune responses in cancer and infectious diseases, while their progressive loss is associated with T-cell exhaustion, senescence and anergy. Consequently, strategies that enhance the multifunctional status of T cells are a key for immunotherapy. Dendritic cells (DCs) are professional antigen presenting cells that regulate T-cell functions by providing positive and negative co-stimulatory signals. A key negative regulator of T-cell activity is provided by binding of programmed death-1 (PD-1) receptor on activated T cells, to its ligand PD-L1, expressed on DCs. We investigated the impact of interfering with PD-L1/PD-1 co-stimulation on the multifunctionality of T cells, by expression of the soluble extracellular part of PD-1 (sPD-1) or PD-L1 (sPD-L1) in human monocyte-derived DCs during antigen presentation. Expression, secretion and binding of these soluble molecules after mRNA electroporation were demonstrated. Modification of DCs with sPD-1 or sPD-L1 mRNA resulted in increased levels of the co-stimulatory molecule CD80 and a distinct cytokine profile, characterized by the secretion of IL-10 and TNF-α, respectively. Co-expression in DCs of sPD-1 and sPD-L1 with influenza virus nuclear protein 1 (Flu NP1) stimulated Flu NP1 memory T cells, with a significantly higher number of multifunctional T cells and increased cytokine secretion, while it did not induce regulatory T cells. These data provide a rationale for the inclusion of interfering sPD-1 or sPD-L1 in DC-based immunotherapeutic strategies.

T Lymphocytes Expressing a CD16 Signaling Receptor Exert Antibody-Dependent Cancer Cell Killing

To expand applications for T-cell-based immunotherapy in cancer, we designed a receptor that binds the Fc portion of human immunoglobulins and delivers activation signals. The construct included the high-affinity CD16 (FCGR3A) V158 variant, CD8α hinge, and transmembrane domains, along with signaling domains from CD3ζ and 4-1BB (TNFRSF9), forming a chimeric receptor termed CD16V-BB-ζ. After retrovirus-mediated expression in human T cells, CD16V-BB-ζ bound humanized antibodies with higher affinity than a control receptor containing the more common F158 variant. Engagement of CD16V-BB-ζ provoked T-cell activation, exocytosis of lytic granules, and sustained proliferation, with a mean cell recovery after 4-week coculture with Daudi lymphoma cells and rituximab of nearly 70-fold relative to input cells. In contrast, unbound antibody alone produced no effect. CD16V-BB-ζ T cells specifically killed lymphoma cells and primary chronic lymphocytic leukemia cells in combination with rituximab at a low effector:target ratio, even when assayed on mesenchymal cells. Trastuzumab triggered CD16V-BB-ζ-mediated killing of HER2 (ERBB2)(+) breast and gastric cancer cells; similar results were obtained with an anti-GD2 antibody in neuroblastoma and osteosarcoma cells. Furthermore, coadministration of CD16V-BB-ζ T cells with immunotherapeutic antibodies exerted considerable antitumor activity in vivo. Signaling mediated by 4-1BB-CD3ζ induced higher T-cell activation, proliferation, and cytotoxicity than CD3ζ or FcεRIγ, and the receptor was expressed effectively after mRNA electroporation without viral vectors, facilitating clinical translation. Our results offer preclinical proof of concept for CD16V-BB-ζ as a universal, next-generation chimeric receptor with the potential to augment the efficacy of antibody therapies for cancer.

Anti-CD20 Chimeric Antigen Receptor (CAR) Modified Expanded Natural Killer (NK) Cells Significantly Mediate Burkitt Lymphoma (BL) Regression and Improve Survival In Human BL Xenografted NSG Mice

BackgroundBurkitt lymphoma (BL) is the most common form of non-Hodgkin lymphoma that occurs in children and adolescents (Miles/Cairo, BJH, 2012). The 5-year event-free survival (EFS) of children and adolescents with newly diagnosed BL has been significantly improved but for patients with relapsed/refractory disease, the prognosis is dismal due to chemotherapy resistance (Cairo, et al, J Clin Oncol, 2012; Cairo, et al Blood 2007). Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. We have previously demonstrated that CD20 is expressing in ≥98% of pediatric BL (Perkins/Cairo, Clin Adv Hematol Oncol. 2003) and also reported that expanded Peripheral Blood Natural Killer (exPBNK) Cells electroporated with anti-CD20 Chimeric Antigen Receptor (CAR) mRNA have significant cytotoxicity against CD20+ BL in vitro (Chu/Cairo, et al, ASH, 2012). ObjectiveTo examine the anti-tumor effect of anti-CD20 chimeric antigen receptor (CAR+) modified expanded PBNK cells against disseminated CD20+ Burkitt Lymphoma in vivo using human CD20+ BL xenografted NSG mice. MethodsPBMC were expanded with lethally irradiated K562-mbIL15-41BBL cells for 14 days as we previously reported. CD56+CD3- expanded PBNK (exPBNK) cells were isolated using Miltenyi NK cell isolation kit. Anti-CD20-4-1BB-CD3ζ mRNA (CAR mRNA) was produced using the mMESSAGE mMACHINE T7 Ultra kit. CAR mRNA was nucleofected and CAR expression was detected using anti-mouse IgG, F(ab')2 fragment-specific antibody (Chu & Cairo, et al, ASH, 2012).5x105 Raji cells expressing luciferase (Raji-Luc) were intraperitoneally (i.p.) injected into the NSG mice (6 weeks, Jackson Labs). Raji engraftment and progression was evaluated using the Xenogen IVIS-200 system (Caliper Life Sciences) after i.p. injection of 150mg D-luciferin/kg/mouse. Photons emitted from luciferase-expression cells were quantified using the Living Image software. After Raji-Luc engraftment was verified in mice at day 7, 5x106 anti-CD20 CAR exPBNK or MOCK exPBNK (no anti-CD20 CAR mRNA electroporation) were i.p. injected to each mouse once a week (day 9,16, 23). Control mice received culture medium instead of NK cells. The cumulative luciferase signals were measured weekly to indicate the tumor growth, dissemination and progression.Statistical probability of survival and comparison of survival curves were determined by Kaplan Meier Method and the log-rank test. The results with a P value < 0.05 were deemed statistically significant. ResultsWe found that luciferase signals from Raji-luc were not significantly different between the untreated, mock exPBNK treated, and CAR exPBNK treated groups (p=0.0538) after the first NK injection. However, the luciferase signals measured in the CAR exPBNK treated group were significantly different than that in the control mice (two tailed P= 0.0013) and the mock exPBNK treated mice (two tailed P= 0.0229) after the second NK injection. After the third NK injection, the luciferase signals measured in the CAR exPBNK treated group were also significantly different than that in the control mice (two tailed P= 0.0173) and the mock exPBNK treated mice (two tailed P= 0.0256) (Fig. 1A). But luciferase signals were not significantly different between the untreated mice and the mock exPBNK treated mice (two tailed P= 0.0729).Consistent with the reduced luciferase signals, the CAR exPBNK treated mice had significantly extended survival time with median 40 days compared to the untreated mice (29 days, P<0.001) and the mock exPBNK treated mice (30 days, P<0.001) (Fig. 1B). There was no significant difference of survival between the untreated mice and the mock exPBNK treated mice. ConclusionMultiple injections of anti-CD20 CAR mRNA electroporated exPBNK cells can significantly mediate disseminated Raji tumor regression in Raji xenografted NSG mice compared to untreated and mock exPBNK treated mice. Furthermore, anti-CD20 CAR exPBNK significantly increased the survival of Raji xenografted mice. These results indicate therapeutic potential of multiple injections of anti-CD20 CAR mRNA modified exPBNK cells against BL in patients with chemo-immunotherapy resistance. Future directions include examining the anti-tumor activity of CAR+ exPBNK against rituximab/chemotherapy resistant BL in xenografted NSG mice. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Langerhans-type and monocyte-derived human dendritic cells have different susceptibilities to mRNA electroporation with distinct effects on maturation and activation: implications for immunogenicity in dendritic cell-based immunotherapy

BackgroundmRNA electroporation of dendritic cells (DCs) facilitates processing and presentation of multiple peptides derived from whole antigen, tailored to different HLA molecules. Clinical responses to electroporated moDC vaccines, however, have been suboptimal. Human Langerhans-type DCs (LCs) are the most potent conventional DC subtype for inducing CD8+ cytotoxic T lymphocytes (CTLs) in vitro. We recently demonstrated that Wilms’ tumor 1 (WT1) mRNA-electroporated LCs are superior to moDCs as stimulators of tumor antigen-specific CD8+ CTLs, even though they are comparable stimulators of allogeneic T cell proliferative responses. A detailed comparative evaluation of the effects of mRNA electroporation on LCs versus moDCs, however, is needed.MethodsImmature and partially-matured human moDCs and LCs electroporated with mRNA were compared for transfection efficiency, phenotypic changes, viability, retention of transgene expression after cryopreservation, and immunogenicity. Student t test was used for each pairwise comparison. One-way analysis of variance was used for multiple group comparisons.ResultsTransfection efficiency after electroporation with enhanced green fluorescent protein (eGFP) mRNA was higher for immature than for partially-matured moDCs. In contrast, transfection efficiency was higher for partially-matured than for immature LCs, with the additional benefit that electroporation itself increased maturation and activation of CD83+HLA-DRbright LCs but not moDCs. Electroporation did not impair final maturation and activation of either DC subtype, after which both mRNA-electroporated LCs and moDCs were functionally similar in stimulating allogeneic T cell proliferation, a standard assay of DC immunogenicity.ConclusionsThese findings support mRNA electroporation of DCs, and in particular LCs, as an effective non-viral method to stimulate specific, potent CD8+ CTL responses. The differences between LCs and moDCs regarding this form of antigen-loading have important implications for DC-based immunotherapies.

Use of mRNA for vaccination or gene therapy: delivery route determines the verdict (P4316)

Abstract The use of DNA and viral vector vaccines for the induction of cellular immune responses is increasingly gaining interest. However, concerns have been raised regarding the safety of these immunization strategies. Due to their lack of genome integration, mRNA-based vaccines have emerged as a promising alternative. In this study we evaluated the potency of antigen-encoding mRNA either delivered via complexation with the cationic lipid DOTAP/DOPE, or delivered via in vivo electroporation as a novel vaccination approach. By using luciferase-encoding mRNA we showed that in vivo electroporation of mRNA induces high levels of luciferase at the injection site, while protein expression in mice injected with DOTAP/DOPE complexed mRNA was barely above background levels. Despite the high levels of protein expression, in vivo electroporation of mRNA failed to elicit immune responses against the encoded antigen. In contrast, lipid-based delivery of mRNA induced antigen-specific, functional T cell responses resulting in specific killing of peptide-pulsed cells and the induction of humoral responses. In summary, in vivo electroporation of antigen-encoding mRNA induces high levels of protein expression but no immune responses, thus presenting an ideal tool for mRNA-based gene therapy, while lipid-based delivery offers advantages for vaccination purposes due to the induction of low protein levels but strong antigen-specific cellular immunity.

183. Electroporation of ZFN mRNA Enables Efficient CCR5 Gene Disruption in Mobilized Blood Hematopoietic Stem Cells at Clinical Scale

183. Electroporation of ZFN mRNA Enables Efficient CCR5 Gene Disruption in Mobilized Blood Hematopoietic Stem Cells at Clinical Scale

Abstract 3972: Carpet bombing with CARs: Influence of dose, timing, lymphodepletion and costimulatory domain on efficacy of mRNA engineered anti-CD19 Chimeric Antigen Receptor T cells in preclinical xenograft models.

Abstract T cells engineered to express chimeric antigen receptors (CARs), consisting of an anti-tumor targeting domain fused to Signal 1 and 2 activation domains, are a promising approach to cell therapy. We compared T cells engineered to stably express CARs using integrating viral vectors with T cells transiently expressing CARs via direct transduction with non-integrating mRNA. In early clinical testing, this CAR design using lentiviral transduction has been tested in patients with advanced CLL and relapsed ALL. Dramatic clinical responses have been observed in these refractory patients, speaking to the power of this approach. Transient expression has several advantages: i) short-term expression may allow initial clinical testing while limiting toxicity, and ii) RNA is easier to manufacture and modify, expediting manufacturing and scaling to GMP of changing CAR designs, allowing rapid iterative testing of and improvements to CAR design. To evaluate the in vivo efficacy of mRNA CAR+T cells compared to permanently modified (lentiviral transduction) T cells, we used xenograft models of human ALL (Nalm-6 and a primary patient leukemia, both CD19+) and high-level human T cell engraftment. NSG mice were injected with ALL at a dose producing 100% BM engraftment by 5d with 100% mortality by 25d (Nalm-6) or 36d (Primary). T cells were expanded ex vivo using CD3/CD28 beads and underwent lentiviral gene transfer or mRNA electroporation. Short-term efficacy of a single dose of mRNA vs. lentiviral CAR+ T cells was similar, and unlike permanently modified CAR T cells the costimulatory domain (CD28, 41BB or both) in mRNA engineered cells had no effect on efficacy in this model. Interval lymphodepletion with cyclophosphamide (CTX, 60mg/kg1) significantly improved efficacy with subsequent infusions of RNA CAR T cells. Neither CTX nor untargeted repeat CAR T cells infusions alone had an effect on leukemia burden or survival. While lentiviral CAR+ T cells persisted and were able to demonstrate a memory response on leukemia rechallenge, median overall survival was not different between repeat mRNA CARs (80d) and a single dose of lentiviral transduced CARs (77d). Importantly, both models produced long term (&amp;gt;120 days) survivors, suggesting that repeat infusion of RNA CAR T cells with interval lymphodepletion may approximate the efficacy of permanently modified T cells in this liquid tumor model. In our clinical experience with permanently modified cells, disease clearance happens over 14-21 days, which correlates with the window of efficacy of mRNA CARs. This suggests that though they do not persist, mRNA CARs may have significant potential for clinical efficacy. These pre-clinical results demonstrate an important advance in T cell engineering, and are leading directly to clinical trials testing mRNA CAR CTLs. Citation Format: David M. Barrett, Yangbing Zhao, Xiaojun Liu, Shuguang Jiang, Gregor S D Reid, Alix E. Seif, Carl H. June, Stephan A. Grupp. Carpet bombing with CARs: Influence of dose, timing, lymphodepletion and costimulatory domain on efficacy of mRNA engineered anti-CD19 Chimeric Antigen Receptor T cells in preclinical xenograft models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3972. doi:10.1158/1538-7445.AM2013-3972

Design of an Optimized Wilms’ Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo

Tumor antigen–encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K.