Field Of Cell Therapy Research Articles

FV Vectors as Alternative Gene Vehicles for Gene Transfer in HSCs.

Hematopoietic Stem Cells (HSCs) are a unique population of cells, capable of reconstituting the blood system of an organism through orchestrated self-renewal and differentiation. They play a pivotal role in stem cell therapies, both autologous and allogeneic. In the field of gene and cell therapy, HSCs, genetically modified or otherwise, are used to alleviate or correct a genetic defect. In this concise review, we discuss the use of SFVpsc_huHSRV.13, formerly known as Prototype Foamy Viral (PFV or FV) vectors, as vehicles for gene delivery in HSCs. We present the properties of the FV vectors that make them ideal for HSC delivery vehicles, we review their record in HSC gene marking studies and their potential as therapeutic vectors for monogenic disorders in preclinical animal models. FVs are a safe and efficient tool for delivering genes in HSCs compared to other retroviral gene delivery systems. Novel technological advancements in their production and purification in closed systems, have allowed their production under cGMP compliant conditions. It may only be a matter of time before they find their way into the clinic.

Accelerating the development of innovative cellular therapy products for the treatment of cancer

The field of cell therapy is rapidly emerging as a priority area for oncology research and drug development. Currently, two chimeric antigen receptor T-cell therapies are approved by the US Food and Drug Administration and other agencies worldwide for two types of hematologic cancers. To facilitate the development of these therapies for patients with life-threatening cancers with limited or no therapeutic options, science- and risk-based approaches will be critical to mitigating and balancing any potential risk associated with either early clinical research or more flexible manufacturing paradigms. Friends of Cancer Research and the Parker Institute for Cancer Immunotherapy convened an expert group of stakeholders to develop specific strategies and proposals for regulatory opportunities to accelerate the development of cell therapies as promising new therapeutics. This meeting took place in Washington, DC on May 17, 2019. As academia and industry expand research efforts and cellular product development pipelines, this report summarizes opportunities to accelerate entry into the clinic for exploratory studies and optimization of cell products through manufacturing improvements for these promising new therapies.

Adoptive Cell Therapy-Harnessing Antigen-Specific T Cells to Target Solid Tumours.

In recent years, much research has been focused on the field of adoptive cell therapies (ACT) that use native or genetically modified T cells as therapeutic tools. Immunotherapy with T cells expressing chimeric antigen receptors (CARs) demonstrated great success in the treatment of haematologic malignancies, whereas adoptive transfer of autologous tumour infiltrating lymphocytes (TILs) proved to be highly effective in metastatic melanoma. These encouraging results initiated many studies where ACT was tested as a treatment for various solid tumours. In this review, we provide an overview of the challenges of T cell-based immunotherapies of solid tumours. We describe alternative approaches for choosing the most efficient T cells for cancer treatment in terms of their tumour-specificity and phenotype. Finally, we present strategies for improvement of anti-tumour potential of T cells, including combination therapies.

A Fast and Sensitive Luciferase-based Assay for Antibody Engineering and Design of Chimeric Antigen Receptors

Success of immunotherapeutic approaches using genetically engineered antibodies and T cells modified with chimeric antigen receptors (CARs) depends, among other things, on the selection of antigen binding domains with desirable expression and binding characteristics. We developed a luciferase-based assay, termed Malibu-Glo Assay, which streamlines the process of optimization of an antigen binding domain with desirable properties and allows the sensitive detection of tumor antigens. The assay involves a recombinant immunoconjugate, termed Malibu-Glo reagent, comprising an immunoglobulin or a non-immunoglobulin based antigen binding domain genetically linked to a marine luciferase. Malibu-Glo reagent can be conveniently produced in mammalian cells as a secreted protein that retains the functional activity of both the antigen binding domain and the luciferase. Moreover, crude supernatant containing the secreted Malibu-Glo reagent can directly be used for detection of cell surface antigens obviating the laborious steps of protein purification and labeling. We further demonstrate the utility of Malibu-Glo assay for the selection of optimal single chain fragment variables (scFvs) with desired affinity characteristics for incorporation into CARs. In summary, Malibu-Glo assay is a fast, simple, sensitive, specific and economical assay for antigen detection with multiple applications in the fields of antibody engineering, antibody humanization and CAR-T cell therapy.

Biomaterials-Induced Stem Cells Specific Differentiation Into Intervertebral Disc Lineage Cells.

Stem cell therapy, which promotes stem cells differentiation toward specialized cell types, increases the resident population and production of extracellular matrix, and can be used to achieve intervertebral disc (IVD) repair, has drawn great attention for the development of IVD-regenerating materials. Many materials that have been reported in IVD repair have the ability to promote stem cells differentiation. However, due to the limitations of mechanical properties, immunogenicity and uncontrollable deviations in the induction of stem cells differentiation, there are few materials that can currently be translated into clinical applications. In addition to the favorable mechanical properties and biocompatibility of IVD materials, maintaining stem cells activity in the local niche and increasing the ability of stem cells to differentiate into nucleus pulposus (NP) and annulus fibrosus (AF) cells are the basis for promoting the application of IVD-regenerating materials in clinical practice. The purpose of this review is to summarize IVD-regenerating materials that focus on stem cells strategies, analyze the properties of these materials that affect the differentiation of stem cells into IVD-like cells, and then present the limitations of currently used disc materials in the field of stem cell therapy and future research perspectives.

The in vitro inertial positions and viability of cells in suspension under different in vivo flow conditions

The influence of Poiseuille flow on cell viability has applications in the areas of cancer metastasis, lab-on-a-chip devices and flow cytometry. Indeed, retaining cell viability is important in the emerging field of cell therapy as cells need to be returned to patients’ bodies. Despite this, it is unclear how this fundamental fluid regime affects cell viability. This study investigated the influence that varying flow rate, and the corresponding wall shear stress (τw) has on the viability and inertial positions of circulating cells in laminar pipe flow. The viability of two representative cell lines under different shear stresses in two different systems were investigated while particle streak imaging was used to determine their inertial positions. It was found that peristaltic pumps have a negative effect on cell viability in comparison to syringe pumps. Increasing shear stress in a cone and plate above 3 Pa caused an increase in cell death, however, τw as high as 10 Pa in circulation has little to no effect on cell viability. Inertial lift forces that move cells towards the centre of the channel protect them from experiencing detrimental levels of τw, indicating that τw in Poiseuille flow is not a good predictor of cell viability during advection.

CAR T-cell product performance in haematological malignancies before and after marketing authorisation.

Chimeric antigen receptor (CAR) T cells represent a potent new approach to treat haematological malignancies. Two CAR T-cell therapies, tisagenlecleucel and axicabtagene ciloleucel, have been approved in Europe and the USA, as well as several other countries, for the treatment of leukaemia and lymphoma. These approvals marked a major milestone in the field of cell and gene therapies. However, the clinical development and regulatory evaluation of these innovative therapies faced several challenges that are considered important lessons learned for future similar products. Here, we examine the products' non-clinical and clinical data packages to outline the challenges encountered during the regulatory evaluation process in Europe, and to provide an update on their performance after authorisation.

Rapid Delivery of Nanobodies/VHHs into Living Cells via Expressing In Vitro-Transcribed mRNA

Intracellular antigen labeling and manipulation by antibodies have been long-thought goals in the field of cell research and therapy. However, a central limitation for this application is that antibodies are not able to penetrate into the cytosol of living cells. Taking advantages of small sizes and unique structures of the single-domain antibodies, here, we presented a novel approach to rapidly deliver the nanobody/variable domain of heavy chain of heavy-chain antibody (VHH) into living cells via introducing its coding mRNA, which was generated by in vitro transcription. We demonstrated that actin-green fluorescent proteins (GFP) and Golgi-GFP can be recognized by the anti-GFP nanobody/VHH, vimentin can be recognized by the anti-vimentin nanobody/VHH, and histone deacetylase 6 (HDAC6) can be recognized by the anti-HDAC6 nanobody/VHH, respectively. We found that the anti-GFP nanobody expressed via in vitro-transcribed (IVT) mRNA can be detected in 3 h and degraded in 48 h after transfection, whereas the nanobody expressed via plasmid DNA, was not detected until 24 h after transfection. As a result, it is effective in delivering the nanobody through expressing the nanobody/VHH in living cells from its coding mRNA.

12th Post-ECTRIMS Meeting: review of the novelties from the 2019 ECTRIMS Congress (II).

Like every year, after the ECTRIMS Congress, renowned Spanish neurologists who are experts in multiple sclerosis presented the main novelties in research in this field at the Post-ECTRIMS Meeting. To summarise the content presented at the 12th edition of the Post-ECTRIMS Meeting, which took place in September 2019 in Sevilla and is presented in two parts. In this second part, the most recent evidence on the use of disease-modifying treatments during pregnancy is presented. Details are provided concerning the results of phase 3 clinical trials conducted to evaluate the efficacy and safety of two potential disease-modifying treatments for relapsing-remitting multiple sclerosis: ponesimod and ofatumumab. For the progressive forms, both available disease modifying treatments and others still in the research phase are reviewed. In the field of stem cell therapies, the article includes the results of the only clinical trial carried out to date comparing patients with relapsing-remitting multiple sclerosis treated with autologous haematopoietic stem cell transplantation and those treated with disease-modifying therapies. There are no important developments as regards symptomatic treatments, although the European Academy of Neurology has published a guide on palliative care. The various sources of information that collect pharmacovigilance data in the post-marketing setting are reviewed. Patients diagnosed in recent years tend to have less severe multiple sclerosis, probably due to the fact that it is diagnosed in its milder stages together with the steady increase in the number of treatments available.

MRNA-Based Reprogramming Under Xeno-Free and Feeder-Free Conditions.

Reprogramming somatic cells into induced pluripotent stem cells (iPSC) has provided a gateway for many novel discoveries in the field of tissue engineering, regenerative medicine and cell therapy. The need for an efficient, less laborious and fast reprogramming protocol under xeno-free, feeder-free and chemically defined conditions has never been greater. Here we describe a novel approach to reprogramming using the StemRNA 3rd Gen Reprogramming Kit (ReproCELL) which encompasses non-modified microRNAs (NM-miRNA), non-modified E3, K3, B18R RNAs (EKB NM-RNA) and non-modified mRNAs for six crucial transcription factors (OSKMNL NM-RNA).

Stem cell research and therapy in India: General awareness for the public and stem cell therapy providers

The rapidly evolving field of stem cell research and therapy, along with changing regulations requires clarity both for stakeholders and patients who take recourse as a last resort. If one was to do a search for a paper on public awareness in this area, there are very few, more so for India. The main objective of this article is to help the lay public and also those interested in providing stem cell therapy understand the current rules and requirements in India. This article provides important information in a concise, to the point and nontechnical way.

Natural polymeric nanoparticles as a non-invasive probe for mesenchymal stem cell labelling

Non-invasive tracking of stem cells after transplant is necessary for cell therapy and tissue engineering field. Herein, we introduce natural and biodegradable nanoparticle to develop a highly efficient nanoprobe with the ability to penetrate the stem cell for tracking. Based on the use of (Gd3+) to label stem cells for magnetic resonance imaging (MRI) we synthesized nanoparticle-containing Gd3+. Gd3+ could be used as t 1-weighted MRI contrast agents. In this study, chitosan-alginate nanoparticles were synthesized as a clinical Dotarem® carrier for decreased t 1-weighted. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), and Fourier-transform infrared spectroscopy (FTIR) were utilized for nanoprobe characterization and ICP analysis was performed for Gd3+ concentration measurement. The results illustrate that nanoprobes with spherical shape and with a size of 80 nm without any aggregation were obtained. Relaxivity results suggest that r 1 in the phantom was 12.8 mM−1s−1 per Gd3+ ion, which is 3.5 times larger than that for Dotarem® (r 1 ∼3.6 mM−1s−1 per Gd3+ ion) and this result for synthesized nanoprobe in stem cells 3.56 mM−1s−1 per Gd3+ ion with 2.16 times larger than that for Dotarem® was reported and also enhanced signal in in-vivo imaging was observed. Chitosan-alginate nanoparticles as a novel biocompatible probe for stem cell tracking can be utilized in tissue engineering approach.

Problems and prospects of cell therapy for critical ischaemia of lower limbs

Cell therapy was proposed as a procedure of indirect revascularization for patients with critical ischaemia of lower extremities for whom endovascular and surgical revascularization is impossible. We present herein a review of the state of the art of studies in the field of cell therapy of this cohort of patients. Cell therapy has proved safe, however, the results of studies of efficacy are relatively ambiguous and unconvincing. The number of patients in separately taken clinical trials is minimal. The reviewed studies differed not only by heterogeneity of the cell types used but by the routes of administration of cells (cells were delivered either intramuscularly (predominantly) or intraarterially) and the duration of follow up (time of assessment and duration of follow up varied from 1 month to 2 years). One of the problems became the lack of the routine study of the angiogenic potential of stem cells prior to their clinical application. It is known that the angiogenic activity of multipotent cells of apparently healthy patients may differ from that of patients suffering from atherosclerosis, chronic renal failure, diabetes. It is supposed that treatment with stem cells or precursor cells is more efficient as compared to protein or gene therapy not only owing to direct vasculogenic properties but a paracrine action through excretion of proangiogenic biologically active substances. More studies with larger cohorts are necessary to provide stronger safety and efficacy data on cell therapy. Besides, a promising trend in the field of cellular approaches is modulation of regenerative capability of stem cells, which may help overcome difficulties in understanding the place of cell therapy in therapeutic angiogenesis.

Regenerative treatment of spinal cord injury. Literature review. Part 3

Treating traumatic spinal cord injuries is one of the most complicated and relevant problems in the modern medicine. In the vast majority of cases spinal cord injury (SCI) leads to persistent disability, with medical, social and economic consequences ensuing for the patient, the family and the state. Modern SCI therapy has a very limited effectiveness and does not allow to sufficiently restore the lost functions of central nervous system. Regenerative methods and particularly cell therapy are very promising to effectively treat SCI. The review highlights SCI epidemiological and pathogenetic problems, existing therapy, as well as promising methods of regenerative therapy. We emphasize the results of preclinical and clinical studies in the field of cell therapy. The review is divided into 4 parts. In the part 3, the description of cell therapy methods continues.Conflict of interest. The authors declare no conflict of interest.

Recent Advances in Mono- and Combined Stem Cell Therapies of Stroke in Animal Models and Humans.

Following the failure of acute neuroprotection therapies, major efforts are currently made worldwide to promote neurological recovery and brain plasticity in the subacute and post-acute phases of stroke. Currently, there is hope that stroke recovery might be promoted by cell-based therapies. The field of stem cell therapy for cerebral ischemia has made significant progress in the last five years. A variety of stem cells have been tested in animal models and humans including adipose stem cells, human umbilical cord blood-derived mesenchymal stem cells, human amnion epithelial cells, human placenta amniotic membrane-derived mesenchymal stem cells, adult human pluripotent-like olfactory stem cells, human bone marrow endothelial progenitor cells, electrically-stimulated human neuronal progenitor cells, or induced pluripotent stem cells (iPSCs) of human origin. Combination therapies in animal models include a mix of two or more therapeutic factors consisting of bone marrow stromal cells, exercise and thyroid hormones, endothelial progenitor cells overexpressing the chemokine CXCL12. Mechanisms underlying the beneficial effects of transplanted cells include the “bystander” effects, paracrine mechanisms, or extracellular vesicles-mediated restorative effects. Mitochondria transfer also appears to be a powerful strategy for regenerative processes. Studies in humans are currently limited to a small number of studies using autologous stem cells mainly aimed to assess tolerability and side-effects of human stem cells in the clinic.

Association of Donor-Recipient HLA Matching with Outcome of Unrelated Donor Hematopoietic Stem Cell Transplantation: A Study from the Cellular Therapy and Immunobiology Working Party (CTIWP) of the European Society for Blood and Marrow Transplantation (EBMT)

Association of Donor-Recipient HLA Matching with Outcome of Unrelated Donor Hematopoietic Stem Cell Transplantation: A Study from the Cellular Therapy and Immunobiology Working Party (CTIWP) of the European Society for Blood and Marrow Transplantation (EBMT)

CMV-Specific T Cells Restricted By Shared and Donor, but Not By Host HLA Molecules Reconstitute in the First 180 Days after Allogeneic HSCT and Protect from CMV Reactivation: Results of a Prospective Observational Study

Introduction: Cytomegalovirus (CMV) reactivation and disease are important risk factors after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and strongly affect morbidity and mortality after transplant. CMV-specific T cell reconstitution controls CMV reactivation and protects against serious adverse events but a protective level of CMV-specific T cell response or standardized method for its monitoring have not been yet determined. Methods: We designed a prospective, single-center observational study to assess if the kinetic and quality of CMV specific T-cell reconstitution impact the incidence and severity of CMV reactivations. We have enrolled 84 consecutive patients affected by hematological malignancies receiving allo-HSCT followed by Cyclophosphamide and Rapamycin between December 2017 and February 2019. Here we report preliminary data on the first 61 patients. Patients received allo-HSCT from family (siblings=10; HLA haploidentical=24), unrelated HLA-matched (n= 24) donors or cord blood (CB, n=3). The CMV serostatus of host (H) and donor (D) pairs was: H+/D+(n=40, 65%), H+/D-(n=20, 33%) and H-/D+ (n=1, 2%); H-/D-(7% of the overall transplanted population at our center) were excluded. CMV DNAemia was assessed weekly in whole blood (WB). Absolute numbers of polyclonal and CMV-specific T cells were quantified by flow cytometry using Troucount™Tubes (BD) and Dextramer®CMV-Kit (Immudex), respectively, in the graft and fresh WB at days -7, +30, +45, +60, +90, +120, +150, +180 and +360. Dextramer CMV kit includes reagents for the identification of CMV-specific lymphocytes restricted for several HLA class I molecules: A*01:01/*02:01/*03:01/*24:02 and B*07:02/*08:01/*35:01. These alleles allowed the longitudinal evaluation of 54 out of 61 (89%) patients. Results: At a median follow-up of 226 days post-HSCT, 31 (57%) patients experienced a CMV-related clinically relevant event (CRE, median +63 days), including 8 patients (15%) with CMV disease (median +59 days). Univariate analyses showed that the incidence of CMV clinically-relevant reactivation (CRE) was influenced by H/D CMV serostatus (0.90 in H+/D- versus 0.44 in H+/D+pairs, p=0.015) and by previous acute Graft-versus-Host Disease (aGvHD) requiring systemic immunosuppression (0.82 in aGvHD grade II-IV versus 0.52 in aGvHD grade 0-I, p=0.051). The disease status at transplant, the donor type (HLA-matched versus HLA-haploidentical/CB donors), donor's or host's age did not significantly affect the probability to develop CRE. For each time-point, we compared the absolute number of CMV-specific lymphocytes in patients experiencing or not a subsequent CRE. Our data demonstrate that higher levels of CMV-specific CD8+T cells in the donor apheresis and at +45 days after allo-HSCT are associated with reduced risk of subsequent CRE (median CMV-specific CD8+cells/kg in the apheresis=5x103in CRE-positive patients (CRE+) and 5x105in CRE-negative patients (CRE-), p=0.012; median CMV-specific CD8+at +45 days=0.14 cells/μL in CRE+and 1.21 cells/μL in CRE-, p=0.034). Furthermore, patients with any Dextramer positivity at +45 days displayed a lower incidence of CRE compared with subjects who were negative (CRE probability: 0.5 vs 1.0, p=0.003). Conversely, the absolute number of neither polyclonal CD3+CD8+T lymphocytes nor total CD3+T cells correlate with subsequent CRE. Taking advantage of the HLA mismatched-HSCT setting, we then dissected CMV-specific T-cell response according to HLA restriction elements (H/D=shared n=45, D-restricted n=14, H-restricted n=11). In H+/D+pairs, we observed a fast and similar kinetic of reconstitution of CMV-specific lymphocytes restricted by H/D and D HLAs. Conversely, in H+/D-pairs, we detected only CMV-specific CD8+lymphocytes restricted for H/D haplotypes. Host-restricted cells remained undetectable for the first 180 days after HSCT. Conclusion: Early after allo-HSCT and in the donor apheresis, the level of CMV-specific CD8+T cells measured by Dextramer staining differs in patients experiencing or not subsequent CRE. Furthermore, our findings indicate that CMV reactivations can prime H/D-restricted T cells presumably educated in the donor thymus; conversely, D- and H-restricted donor-derived lymphocytes have not yet undergone neither cross-priming nor thymic education respectively. Disclosures Brix: Immudex: Employment. Bonini:Kite/Gilead: Consultancy; Intellia Therapeutics: Consultancy; Intellia Therapeutics: Research Funding; Novartis: Consultancy; GSK: Consultancy; Allogene: Consultancy; Molmed: Consultancy; TxCell: Consultancy; -: Patents & Royalties: Adoptive T cell therapy field.

Inducible MyD88 Signaling Enhances the Anti-AML Activity of BiTE-Secreting ENG T-Cells

Background: Pediatric and adult acute myeloid Leukemia (AML) is a disease with poor prognosis due to its high relapse rate and treatment related mortality. Adoptive immunotherapy has the potential to improve outcomes and CD123 present a promising immunotherapy target for AML. CD123-specific engager (ENG) T-cells are cells genetically modified to secrete bispecific T-cell engagers (BiTEs) that recognize CD3+ on T-cells and CD123+ on AML. We had previously shown that inclusion of an inducible MyD88/CD40 costimulatory molecule, which is activated by a chemical inducer of dimerization (CID), improved anti-tumor effect against CD123+ targets in vitro. We now wanted to determine, which component of the costimulatory molecule is critical for the improved effector function of CD123-ENG T cells and perform in vivo studies. Methods: We generated a panel of retroviral vectors encoding CD20 as a safety switch, CD123-ENG, and an inducible costimulatory molecule consisting of a myristoylation sequence, FKBP dimerizer domains and one of three signaling costimulatory domains (MyD88 (iM) ,CD40(iC) or MyD88/CD40(iMC)). A vector specific for CD19 (CD20.CD19-ENG.iMC) and non-transduced (NT) T-cells served as controls. We genetically modified T-cells using a retroviral transduction protocol and evaluated their effector function +/- CID. We assessed the effector function of transduced T-cells using flow cytometry, ELISA and luciferase based cytotoxicity assays. We evaluated antitumor activity and persistence in vivo in a xenograft AML model. Results: We successfully generated CD20.CD123-ENG.iM, CD20.CD123-ENG.iC and CD20.CD123-ENG.iMC T-cells. Transduction efficiency ranged from 45 to 70% (±5). The immunophenotype of CD123-ENG T-cells was predominandly CD8+ effector memory. CD20.CD123-ENG T-cells expressing inducible costimulatory molecules secreted higher levels of CD123-specific BiTEs, IL2 and IFNγ in comparison to CD20.CD123-ENG T-cells when cocultured with of MOLM-13 (CD123+) and CID (p<0.005, N=3). In the presence of CID, CD20.CD123-ENG.iM or CD20.CD123-ENG.iMC T-cells were able to sequentially kill AML cells for up to 5 stimulations, as opposed to CD20.CD123 ENG, CD20.CD123 ENG.iC and NT T-cells (n=3, E:T=1:1). We evaluated the in vivo anti-AML activity of ENG T cells using an AML cell line expressing firefly luciferase (MOLM-13.ffluc) to allow for serial bioluminescence imaging. MOLM-13.ffluc bearing mice were treated with one i.v. dose of effector cells (CD20.CD123-ENG, CD20.CD123-ENG iM, CD20.CD123-ENG.iC, CD20.CD123-ENG.iMC or NT T-cells ) and 2 doses/week of CID for 2 weeks (100mg/mice, i.p.). CD20.CD123-ENG.iMC and iM T-cells had potent anti-leukemic activity, resulting in a significant survival advantage in comparison to CD20.CD123-ENG T-cells group (P<0.005, n=10 animals per group). Conclusion: We demonstrate here that inducible MyD88/CD40 costimulation significantly enhances the anti-AML activity of CD123-ENG T cells in vitro and in xenograft models. Mechanistic studies revealed that MyD88 costimulation is critical for the observed benefit. Thus, genetically modifying T-cells to express BiTEs and an inducible MyD88 costimulatory molecule may present a promising cell product for the immunotherapy of CD123+ hematological malignancies. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:TESSA Therapeutics: Other: Research Collaboration; Tidal: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Inmatics: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; EMD Serono: Honoraria; ViraCyte: Consultancy; NHLBI: Research Funding; ASSISI fundation of Memphis: Research Funding; California Institute for Regenerative Medicine: Research Funding; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; America Lebanese Syrian Associated Charities: Research Funding. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties: Patent Applications in the Fields of Cell and Gene Therapy .

Engineering Naturally Occurring CD7 Negative Cells for the Immunotherapy of CD7 Positive Leukemia

Background: CD7 has emerged as a promising target for the adoptive immunotherapy with T-cells expressing chimeric antigen receptors (CAR T-cells) of CD7+ T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). However, expressing CD7 CARs in T-cells results in fratricide due to high expression of CD7 in most T-cells. While investigators have developed strategies to overcome this limitation by additional genetic modifications of CD7 CAR T-cells, the goal of this project was to explore the feasibility of selecting and genetically modifying naturally occurring CD7 negative (CD7-) T cells for the adoptive immunotherapy of CD7+ leukemia. Methods: CD7- T-cells were isolated from PBMCs using a 2-step magnetic bead depletion/selection procedure (CD7 depletion followed by selection of CD3+ T cells from the CD7- fraction). Non-selected T-cells (bulk T-cells), CD7+ and CD7- T cells were activated and transduced with a retroviral vector encoding a second-generation CD7 CAR with a CD28 costimulatory endodomain, and expanded with IL7 and IL15. The effector function of CD7- T-cells expressing CD7 CARs (CD7 CARCD7- T cells) was assessed in vitro as well as in xenograft models. Results: To assess the feasibility of our approach, we first determined the frequency of CD7- T-cells in PBMCs. On average, 4.7 % of T cells were CD7- (range: 2% - 12.3%; N=22), and we successfully selected these cells from bulk PBMCs with a combined CD7 depletion/CD3 selection procedure. We genetically modified CD7-, CD7+ and bulk T cellsto express CD7 CARs (CD7 CARCD7-, CD7 CARCD7+, CD7 CARBulk). Transduction efficiencies ranged from 31% to 75% (± 5%) for each T-cell population. Post transduction, CD7 CARCD7- T-cells did not undergo fratricide and had similar expansion kinetics (N=6, p=ns) in comparison to non-transduced (NT) T-cell cultures (NT CD7-, NT CD7+, NT bulk). In contrast, CD7 CARCD7+or CD7 CARBulk T-cells underwent fratricide and did not expand (N=6, p<0.0001). CD7- T-cells (NT and CD7 CARCD7-) had a predominantly CD4+ effector memory phenotype at day 7 and 14 of culture. To assess the effector function of CD7 CARCD7- T-cells, we co-cultured them with CD7+ T-ALL cell lines (CCRF, MOLT3). CD7 CARCD7- T-cells recognized CD7+ targets in contrast to CD7- targets (BV173, Daudi) as evidenced by significant (N=6, p<0.0001) IFN-γ and IL-2 production. Control CAR T-cells (CD19 CARCD7-) did not recognize CD7+ target cells, confirming specificity. CD7 CARCD7- T-cells also had potent cytolytic activity against CD7+ targets in cytotoxicity assays. To assess in vivo the anti-tumor activity of CD7 CARCD7- T-cells, we used a NSG mouse xenograft model with CCRF cells, genetically modified to express firefly luciferase (CCRF.ffluc) to allow for serial bioluminescence imaging. A single infusion of CD7 CARCD7- T-cells had potent anti-leukemia activity as judged by serial imaging resulting in a significant survival (p<0.003) advantage in comparison to control mice. Conclusion: We have successfully generated CD7 CARCD7- T-cells from peripheral blood CD7- T-cells. CD7 CARCD7- T-cells had a predominantly CD4+ effector memory phenotype, and potent anti-leukemia activity in vitro and in vivo. Thus, naturally occurring CD7- T cells may present a promising T-cell source for the cellular immunotherapy of CD7+ leukemia. Disclosures Langfitt: MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Youngblood:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Gottschalk:NHLBI: Research Funding; America Lebanese Syrian Associated Charities: Research Funding; ASSISI fundation of Memphis: Research Funding; California Institute for Regenerative Medicine: Research Funding; ViraCyte: Consultancy; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; TESSA Therapeutics: Other: Research Collaboration; Tidal: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; EMD Serono: Honoraria; Merck: Consultancy; Inmatics: Membership on an entity's Board of Directors or advisory committees. Velasquez:St. Jude: Patents & Royalties: Patent Applications in the Fields of Cell and Gene Therapy ; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees.

Transgenic Expression of IL15 in CD123-Specific BiTE-Secreting Engager T-Cells Results in Improved Anti-AML Activity

Background: CD123 is frequently expressed on hematologic malignancies including 96-98% of AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) showed a promising approach anti-AML activity, however T-cell persistence was limited. Interleukin-15 (IL15) has emerged as a candidate immunomodulator as it enhances T-cell expansion and persistence, and induces long-lasting memory T-cells. To improve the efficacy and persistence of CD123-ENG T-cells we developed IL15 expressing CD123-ENG T-cells. Here, we report on the characterization and efficacy of IL15-secreting CD123-ENG T cells in vitro and in vivo models of adult AML. Methods/Results: A cDNA encoding IL15 was cloned into retroviral vectors encoding CD123-ENG or CD19-ENG (CD123-ENG.IL15; CD19-ENG.IL15). ENG T-cells were generated from human peripheral blood mononuclear cells (PBMCs) from normal donors or T-cells from AML patients by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells and T-cells expressing CD123 (CD123-ENG T-cells) served as controls. IL15 production of CD19-ENG.IL15 and CD123-ENG.IL15 T cells was confirmed by ELISA (144-159 pg/ml vs 38 and 46 pg/ml of NT and CD123-ENG T cells, p<0.01, n=6). Both CD123-ENG and CD123-ENG.IL15 T-cells recognized CD123+ AML cells as judged by IL2 and interferon γ (IFNγ) production (p<0.01, n=5). In contrast, NT and CD19-ENG.IL15 T-cells did not, confirming specificity. In addition, CD123-ENG.IL15 and CD123-ENG T-cells induced killing of only CD123-positive target cells as well as of primary adult patients' AML blasts in luciferase- or 7AAD-based cytotoxicity assays (p<0.001, n=10). CD123-ENG.IL15 T-cells showed greater cytolytic activity than CD123-ENG T-cells as determined by luciferase activity (p=0.0002, n=3). In a Molm13 AML xenograft model, CD123-ENG.IL15 and CD123-ENG T-cells exhibited potent anti-leukemic activity as judged by bioluminescence imaging. Moreover, CD123-ENG.IL15 T cells had enhanced anti-leukemic activity and greater persistence in BMs, spleens, and livers in comparison to CD123-ENG T cells, resulting in improved anti-AML activity (Figure 1, p<0.01 vs CD123-ENG T-cell group, n=12 per group) and extended survival (Figure 2, p=0.0097 vs CD123-ENG T-cell group). Finally, AML PDX models and ENG T-cells were generated from AML blasts and T-cells from 3 patients with active AML. Infusion of autologous ENG T-cells (1.5x106 cells/mouse, n=7) in AML PDX#6697688 mouse model revealed significant reduction of leukemia burden in the CD123-ENG.IL15 or CD123-ENG T-cells mouse groups but not in the mouse group with NT or CD19-ENG.IL15 T-cells or PBS (p=0.004, n=6-8). We are currently monitoring survival of these PDX models. Conclusion: We here demonstrated that transgenic expression of IL15 in CD123-ENG T-cells results in improved expansion and persistence, and anti-AML activity. These results warrant further exploration of IL15-modified CD123-targeted T-cells as immunotherapy for AML. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:EMD Serono: Honoraria; Inmatics: Membership on an entity's Board of Directors or advisory committees; ASSISI fundation of Memphis: Research Funding; TESSA Therapeutics: Other: Research Collaboration; ViraCyte: Consultancy; NHLBI: Research Funding; America Lebanese Syrian Associated Charities: Research Funding; California Institute for Regenerative Medicine: Research Funding; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; Sanofi: Honoraria; Tidal: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy. Velasquez:St. Jude: Patents & Royalties: Patent Applications in the Fields of Cell and Gene Therapy ; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees. Andreeff:Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstaZeneca: Consultancy; Amgen: Consultancy; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; CPRIT: Research Funding; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding.