Journal Articles

Adoptive T cell therapy

T cell therapies based on tumor infiltrating T lymphocytes and chimeric antigen receptor or T cell receptor engineered T cells

CD28-zeta CAR T Cells Resist TGF-beta Repression through IL-2 Signaling, Which Can Be Mimicked by an Engineered IL-7 Autocrine Loop

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Adoptive cell therapy with chimeric antigen receptor (CAR)-redirected T cells induced spectacular regressions of leukemia and lymphoma, however, failed so far in the treatment of solid tumors. A cause is thought to be T cell repression through TGF-beta, which is massively accumulating in the tumor tissue. Here, we show that T cells with a CD28-zeta CAR, but not with a 4-1BB-zeta CAR, resist TGF-beta-mediated repression. Mechanistically, LCK activation and consequently IL-2 release and autocrine IL-2 receptor signaling mediated TGF-beta resistance; deleting the LCK-binding motif in the CD28 CAR abolished both IL-2 secretion and TGF-beta resistance, while IL-2 add-back restored TGF-beta resistance. Other gamma-cytokines like IL-7 and IL-15 could replace IL-2 in this context. This is demonstrated by engineering IL-2 deficient CD28DeltaLCK-zeta CAR T cells with a hybrid IL-7 receptor to provide IL-2R beta chain signaling upon IL-7 binding. Such modified T cells showed improved CAR T cell activity against TGF-beta(+) tumors. Data draw the concept that an autocrine loop resulting in IL-2R signaling can make CAR T cells more potent in staying active against TGF-beta(+) solid tumors.

Author Info: (1) Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Department I Internal Medicine, University Hospital Cologne, Cologne, Germany. (2) Center for Molecular Medicine

Author Info: (1) Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Department I Internal Medicine, University Hospital Cologne, Cologne, Germany. (2) Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Department I Internal Medicine, University Hospital Cologne, Cologne, Germany. (3) Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Department I Internal Medicine, University Hospital Cologne, Cologne, Germany. (4) Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Department I Internal Medicine, University Hospital Cologne, Cologne, Germany; Regensburg Center for Interventional Immunology (RCI), University Regensburg, Regensburg, Germany; University Medical Center of Regensburg, Regensburg, Germany. Electronic address: hinrich.abken@ukr.de.

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Quantification of B-cell maturation antigen, a target for novel chimeric antigen receptor T-cell therapy in Myeloma

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B-cell maturation antigen (BCMA) is expressed by normal and malignant plasma cells and is targeted via anti-BCMA chimeric antigen receptor T-cell therapy (BCMA CAR T-cell therapy) in plasma cell myeloma (PCM) patients. Surface BCMA expression is required for CAR T-cell binding and killing. We determined the incidence and intensity of expression of BCMA in bone marrow PCM cells using flow cytometry (FC) and immunohistochemistry (IHC). PCM BCMA expression was assessed by FC in 70 patients and in 43 concurrent specimens by IHC. BCMA expression was detected in 94% of patients. FC could assess BCMA expression in all specimens and expression was quantifiable (QuantiBRITE system, BD Biosciences, San Jose, CA) in 89% of cases. Expression was highly variable and could be numerically classified into dim, moderate or bright levels of expression. In the 43 specimens assessed successfully by both IHC and FC, FC showed higher positivity rate (97%) than IHC (72%), indicating that FC is more useful than IHC in detection of BCMA (p=0.002; McNemar's test). We conclude that FC is more sensitive than IHC and can be used to objectively quantify BCMA expression by myeloma cells. IHC is primarily useful when there is significant infiltration of the bone marrow by myeloma and is less sensitive with low numbers of myeloma cells. Furthermore, the ability of FC to differentiate between normal and abnormal plasma cells and to quantify BCMA on these cells, makes it a useful and sensitive tool in screening patients for CAR T-cell therapy and for follow-up post therapy.

Author Info: (1) Flow Cytometry, Laboratory of Pathology, CCR, NCI, NIH, USA; Clinical Pathology Department, Faculty of Medicine, Mansoura University, Egypt. Electronic address: salemda@mail.nih.gov. (2) Hematology, DLM

Author Info: (1) Flow Cytometry, Laboratory of Pathology, CCR, NCI, NIH, USA; Clinical Pathology Department, Faculty of Medicine, Mansoura University, Egypt. Electronic address: salemda@mail.nih.gov. (2) Hematology, DLM, CCR, NCI, NIH, USA. (3) Flow Cytometry, Laboratory of Pathology, CCR, NCI, NIH, USA. (4) Biostatistics and Data Management Section, CCR, NCI, NIH, USA. (5) Biostatistics and Data Management Section, CCR, NCI, NIH, USA. (6) Experimental Transplantation and Immunology Branch, CCR, NCI, NIH, USA. (7) Flow Cytometry, Laboratory of Pathology, CCR, NCI, NIH, USA.

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Hyaluronic acid based low viscosity hydrogel as a novel carrier for Convection Enhanced Delivery of CAR T cells

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Convection Enhanced Delivery (CED) infuses therapeutic agents directly into the intracranial area continuously under pressure. The convection improves the distribution of therapeutics such as those aimed at brain tumors. Although CED successfully delivers small therapeutic agents, this technique fails to effectively deliver cells largely due to cell sedimentation during delivery. To overcome this limitation, we have developed a low viscosity hydrogel (LVHydrogel), which is capable of retaining cells in suspension. In this study, we evaluated whether LVHydrogel can effectively act as a carrier for the CED of tumor-specific chimeric antigen receptor (CAR) T cells. CAR T cells were resuspended in saline or LVHydrogel carriers, loaded into syringes, and passed through the CED system for 5h. CAR T cells submitted to CED were counted and the efficiency of delivery was determined. In addition to delivery, the ability of CAR T cells to migrate and induce cytotoxicity was evaluated. Our studies demonstrate that LVHydrogel is a superior carrier for CED in comparison to saline. The efficiency of cell delivery in saline carrier was only approximately 3-5% of the total cells whereas delivery by the LVHydrogel carrier was much higher, reaching approximately 45-75%. Migration and Cytotoxicity was similar in both carriers in non-infused samples but we found superior cytotoxicity in LVHydrogel group post-infusion. We demonstrate that LVHydrogel, a biodegradable biomaterial which does not cause acute toxicity on preclinical animal models, prevents cellular sedimentation during CED and presents itself as a superior carrier to the current carrier, saline, for the CED of CAR T cells.

Author Info: (1) Duke Brain Tumor Immunotherapy Program, Department of Neurosurgery, Duke University Medical Center, Durham, NC 27710, United States; The Preston Robert Tisch Brain Tumor Center

Author Info: (1) Duke Brain Tumor Immunotherapy Program, Department of Neurosurgery, Duke University Medical Center, Durham, NC 27710, United States; The Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC 27710, United States. (2) Duke Brain Tumor Immunotherapy Program, Department of Neurosurgery, Duke University Medical Center, Durham, NC 27710, United States; The Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC 27710, United States; Department of Pathology, Duke University Medical Center, Durham, NC 27710, United States. (3) Department of Biomedical Engineering, Duke University, Durham, NC 27708, United States; Regeneration Next, Duke University, Durham, NC 27710, United States. (4) Department of Biomedical Engineering, Duke University, Durham, NC 27708, United States. (5) Department of Biostatistics and Bioinformatics, Duke University, Durham, NC 27710, United States. (6) Department of Biostatistics and Bioinformatics, Duke University, Durham, NC 27710, United States. (7) Duke Brain Tumor Immunotherapy Program, Department of Neurosurgery, Duke University Medical Center, Durham, NC 27710, United States; The Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC 27710, United States. (8) Duke Brain Tumor Immunotherapy Program, Department of Neurosurgery, Duke University Medical Center, Durham, NC 27710, United States; The Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC 27710, United States. (9) Department of Pathology, Duke University Medical Center, Durham, NC 27710, United States. (10) Duke Brain Tumor Immunotherapy Program, Department of Neurosurgery, Duke University Medical Center, Durham, NC 27710, United States; The Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC 27710, United States. (11) Duke Brain Tumor Immunotherapy Program, Department of Neurosurgery, Duke University Medical Center, Durham, NC 27710, United States; The Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC 27710, United States; Department of Pathology, Duke University Medical Center, Durham, NC 27710, United States. (12) Duke Brain Tumor Immunotherapy Program, Department of Neurosurgery, Duke University Medical Center, Durham, NC 27710, United States; The Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC 27710, United States; Department of Pathology, Duke University Medical Center, Durham, NC 27710, United States. Electronic address: john.sampson@duke.edu.

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Anti-tumor activity of CAR-T cells targeting the intracellular oncoprotein WT1 can be enhanced by vaccination

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The recent success of chimeric antigen receptor (CAR)-T cell therapy for treatment of hematologic malignancies supports further development of treatments for both liquid and solid tumors. However, expansion of CAR-T cell therapy is limited by the availability of surface antigens specific for the tumor while sparing normal cells. There is a rich diversity of tumor antigens from intracellularly expressed proteins that current and conventional CAR-T cells are unable to target. Furthermore, adoptively transferred T cells often suffer from exhaustion and insufficient expansion, in part, due to the immunosuppressive mechanisms operating in tumor-bearing hosts. Therefore, it is necessary to develop means to further activate and expand those CAR-T cells in vivo. The Wilms tumor 1 (WT1) is an intracellular oncogenic transcription factor that is an attractive target for cancer immunotherapy because of its over-expression in a wide range of leukemias and solid tumors, and a low level of expression in normal adult tissues. In the present study, we developed CAR-T cells consisting of a scFv specific to the WT1235-243/HLA-A*2402 complex. The therapeutic efficacy of our CAR-T cells was demonstrated in a xenograft model, which was further enhanced by vaccination with DCs loaded with the corresponding antigen. This enhanced efficacy was mediated, at least partly, by the expansion and activation of CAR-T cells. CAR-T cells shown in the present study not only demonstrate the potential to expands the range of targets available to CAR-T cells, but also provide a proof-of-concept that efficacy of CAR-T cells targeting peptide/MHC can be boosted by vaccination.

Author Info: (1) Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan. (2) Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie

Author Info: (1) Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan. (2) Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan. (3) Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan. (4) Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan. (5) Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan. (6) Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan. (7) Takara Bio Inc., Shiga, Japan. (8) Takara Bio Inc., Shiga, Japan. (9) Takara Bio Inc., Shiga, Japan. (10) Department of Oncology, Nagasaki University Graduate School of Biomedical Sciences, Japan. (11) Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Hokkaido, Japan. (12) Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime University Graduate School of Medicine, Ehime, Japan. (13) Department of Hematology and Oncology, Fujita Health University, Toyoake, Aichi, Japan. (14) Department of Cellular and Molecular Immunology, Mie University Graduate School of Medicine, Mie, Japan; katotaku@doc.medic.mie-u.ac.jp. (15) Center for Comprehensive Cancer Immunotherapy, Mie University, Mie, Japan.

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Efficient Enrichment of Gene-Modified Primary T Cells via CCR5-Targeted Integration of Mutant Dihydrofolate Reductase

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Targeted gene therapy strategies utilizing homology-driven repair (HDR) allow for greater control over transgene integration site, copy number, and expression-significant advantages over traditional vector-mediated gene therapy with random genome integration. However, the relatively low efficiency of HDR-based strategies limits their clinical application. Here, we used HDR to knock in a mutant dihydrofolate reductase (mDHFR) selection gene at the gene-edited CCR5 locus in primary human CD4(+) T cells and selected for mDHFR-modified cells in the presence of methotrexate (MTX). Cells were transfected with CCR5-megaTAL nuclease mRNA and transduced with adeno-associated virus containing an mDHFR donor template flanked by CCR5 homology arms, leading to up to 40% targeted gene insertion. Clinically relevant concentrations of MTX led to a greater than 5-fold enrichment for mDHFR-modified cells, which maintained a diverse TCR repertoire over the course of expansion and drug selection. Our results demonstrate that mDHFR/MTX-based selection can be used to enrich for gene-modified T cells ex vivo, paving the way for analogous approaches to increase the percentage of HIV-resistant, autologous CD4(+) T cells infused into HIV(+) patients, and/or for in vivo selection of gene-edited T cells for the treatment of cancer.

Author Info: (1) Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. (2) Center for Immunity and Immunotherapies and Program for Cell

Author Info: (1) Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. (2) Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA, USA. (3) Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA, USA. (4) Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. (5) bluebird bio, Inc, Seattle, WA, USA. (6) Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA, USA. Department of Pediatrics, University of Washington, Seattle, WA, USA. Department of Immunology, University of Washington, Seattle, WA, USA. (7) Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. Department of Medicine, University of Washington, Seattle, WA, USA. Department of Pathology, University of Washington, Seattle, WA, USA. (8) Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. Department of Medicine, University of Washington, Seattle, WA, USA.

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Ex vivo expanded tumour-infiltrating lymphocytes from ovarian cancer patients release anti-tumour cytokines in response to autologous primary ovarian cancer cells

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Epithelial ovarian cancer (EOC) is the leading cause of gynaecological cancer-related death in Europe. Although most patients achieve an initial complete response with first-line treatment, recurrence occurs in more than 80% of cases. Thus, there is a clear unmet need for novel second-line treatments. EOC is frequently infiltrated with T lymphocytes, the presence of which has been shown to be associated with improved clinical outcomes. Adoptive T-cell therapy (ACT) using ex vivo-expanded tumour-infiltrating lymphocytes (TILs) has shown remarkable efficacy in other immunogenic tumours, and may represent a promising therapeutic strategy for EOC. In this preclinical study, we investigated the efficacy of using anti-CD3/anti-CD28 magnetic beads and IL-2 to expand TILs from freshly resected ovarian tumours. TILs were expanded for up to 3 weeks, and then subjected to a rapid-expansion protocol (REP) using irradiated feeder cells. Tumours were collected from 45 patients with EOC and TILs were successfully expanded from 89.7% of biopsies. Expanded CD4(+) and CD8(+) subsets demonstrated features associated with memory phenotypes, and had significantly higher expression of key activation and functional markers than unexpanded TILs. Expanded TILs produced anti-tumour cytokines when co-cultured with autologous tumour cells, inferring tumour cytotoxicity. Our findings demonstrate that it is possible to re-activate and expand tumour-reactive T cells from ovarian tumours. This presents a promising immunotherapy that could be used sequentially or in combination with current therapeutic strategies.

Author Info: (1) Gynaecological Oncology, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester

Author Info: (1) Gynaecological Oncology, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK. St Mary's Hospital, Central Manchester NHS Foundation Trust, Manchester Academic Health Science Centre, Level 5, Research Floor, Oxford Road, Manchester, M13 9WL, UK. Clinical and Experimental Immunotherapy, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, Manchester Cancer Research Centre, University of Manchester, Wilmslow Road, Manchester, UK. (2) Gynaecological Oncology, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK. St Mary's Hospital, Central Manchester NHS Foundation Trust, Manchester Academic Health Science Centre, Level 5, Research Floor, Oxford Road, Manchester, M13 9WL, UK. (3) Targeted Therapy Group, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, Manchester Cancer Research Centre, University of Manchester, Wilmslow Road, Manchester, UK. (4) Clinical and Experimental Immunotherapy, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, Manchester Cancer Research Centre, University of Manchester, Wilmslow Road, Manchester, UK. (5) Clinical and Experimental Immunotherapy, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, Manchester Cancer Research Centre, University of Manchester, Wilmslow Road, Manchester, UK. (6) Gynaecological Oncology, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK. richard.edmondson@manchester.ac.uk. St Mary's Hospital, Central Manchester NHS Foundation Trust, Manchester Academic Health Science Centre, Level 5, Research Floor, Oxford Road, Manchester, M13 9WL, UK. richard.edmondson@manchester.ac.uk.

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Bacteria-free minicircle DNA system to generate integration-free CAR-T cells

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BACKGROUND: Chimeric antigen receptor T (CAR-T) cells engineered with lentiviral and retroviral vectors have been successfully applied to treat patients with B cell malignancy. However, viral integration in T cells has the potential risk of mutagenesis, and viral vector production demands effort and is costly. Using non-integrative episomal vector such as minicircle vector to generate integration-free CAR-T cells is an attractive option. METHODS AND RESULTS: We established a novel method to generate minicircle vector within a few hours using simple molecular biology techniques. Since no bacteria is involved, we named these vectors bacteria-free (BF) minicircle. In comparison with plasmids, BF minicircle vector enabled higher transgene expression and improved cell viability in human cell line, stem cells and primary T cells. Using BF minicircle vector, we generated integration-free CAR-T cells, which eliminated cancer cells efficiently both in vitro and in vivo. CONCLUSION: BF minicircle vector will be useful in basic research as well as in clinical applications such as CAR-T and gene therapy. Although the transgene expression of minicircle vector lasts apparently shorter than that of insertional lentivirus, multiple rounds of BF minicircle CAR-T cell infusion could eliminate cancer cells efficiently. On the other hand, a relatively shorter CAR-T cell persistence provides an opportunity to avoid serious side effects such as cytokine storm or on-target off-tumour toxicity.

Author Info: (1) School of Life Sciences, University of Science and Technology of China, Hefei, China. State Key Laboratory of Stem Cell and Reproductive Biology, Institute of

Author Info: (1) School of Life Sciences, University of Science and Technology of China, Hefei, China. State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, China. (2) State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, China. (3) Northwest Agriculture and Forestry University, Yangling, China. (4) State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, China. University of the Chinese Academy of Sciences, Beijing, China. (5) State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, China. University of the Chinese Academy of Sciences, Beijing, China. (6) Beijing Cord Blood Bank, Beijing, China. (7) State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, China. University of the Chinese Academy of Sciences, Beijing, China.

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Reducing Ex Vivo Culture Improves the Anti-leukemic Activity of Chimeric Antigen Receptor (CAR)-T Cells

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The success of chimeric antigen receptor (CAR)-mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells ex vivo for 9-14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of ex vivo culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR targeting CD19 (CART19) exhibited less differentiation and enhanced effector function in vitro when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the anti-leukemic activity inversely correlated with ex vivo culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale cGMP-compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies.

Author Info: (1) Department of Pathology and Laboratory Medicine, Center for Cellular Immunotherapies. (2) Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania

Author Info: (1) Department of Pathology and Laboratory Medicine, Center for Cellular Immunotherapies. (2) Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania. (3) Department of Pathology and Laboratory Medicine, Center for Cellular Immunotherapies. (4) Abramson Cancer Center, University of Pennsylvania. (5) Department of Pathology and Laboratory Medicine, Center for Cellular Immunotherapies. (6) Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine. (7) Division of Oncology, Department of Pediatrics, Children's Hospital of Philadelphia and Perelman School of Medicine at the University of Pennsylvania. (8) Department of Pathology and Laboratory Medicine, Center for Cellular Immunotherapies. (9) Pathology and Laboratory Medicine, Center for Cellular Immunotherapies, University of Pennsylvania. (10) Department of Pathology and Laboratory Medicine, Center for Cellular Immunotherapies. (11) Department of Pathology and Laboratory Medicine, Center for Cellular Immunotherapies. (12) Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania. (13) Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania. (14) Division of Oncology, Department of Pediatrics, Children's Hospital of Philadelphia and Perelman School of Medicine at the University of Pennsylvania. (15) Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania. (16) Pathology and Laboratory Medicine, University of Pennsylvania. (17) Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania milone@pennmedicine.upenn.edu.

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Adenovirus Coding for Interleukin-2 and Tumor Necrosis Factor Alpha Replaces Lymphodepleting Chemotherapy in Adoptive T Cell Therapy

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Lymphodepleting preconditioning with high-dose chemotherapy is commonly used to increase the clinical efficacy of adoptive T cell therapy (ACT) strategies, however, with severe toxicity for patients. Conversely, oncolytic adenoviruses are safe and, when engineered to express interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-alpha), they can achieve antitumor immunomodulatory effects similar to lymphodepletion. Therefore, we compare the safety and efficacy of such adenoviruses with a cyclophosphamide- and fludarabine-containing lymphodepleting regimen in the setting of ACT. Human adenovirus (Ad5/3-E2F-D24-hTNF-alpha-IRES-hIL-2; TILT-123) replication was studied using a Syrian hamster pancreatic tumor model (HapT1) infused with tumor-infiltrating lymphocytes (TILs). Using the oncolytic virus instead of lymphodepletion resulted in superior efficacy and survival. Immune cells responsive to TNF-alpha IL-2 were studied using an immunocompetent mouse melanoma model (B16.OVA) infused with ovalbumin-specific T (OT-I) cells. Here, the adenovirus approach improved tumor control together with increased intratumoral Th1 cytokine levels and infiltration of CD8+ T cells and CD86+ dendritic cells. Similar to humans, lymphodepleting preconditioning caused severe cytopenias, systemic inflammation, and damage to vital organs. Toxicity was minimal in adenovirus- and OT-I-treated mice. These findings demonstrate that ACT can be effectively facilitated by cytokine-coding adenovirus without requiring lymphodepletion, a rationale being clinically investigated.

Author Info: (1) TILT Biotherapeutics Ltd., 00290 Helsinki, Finland; Cancer Gene Therapy Group, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland. (2) TILT Biotherapeutics Ltd., 0029

Author Info: (1) TILT Biotherapeutics Ltd., 00290 Helsinki, Finland; Cancer Gene Therapy Group, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland. (2) TILT Biotherapeutics Ltd., 00290 Helsinki, Finland; Cancer Gene Therapy Group, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland. (3) TILT Biotherapeutics Ltd., 00290 Helsinki, Finland; Cancer Gene Therapy Group, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland. (4) Cancer Gene Therapy Group, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland. (5) TILT Biotherapeutics Ltd., 00290 Helsinki, Finland; Cancer Gene Therapy Group, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland. (6) TILT Biotherapeutics Ltd., 00290 Helsinki, Finland; Cancer Gene Therapy Group, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland. (7) Pathology Unit, Finnish Food Safety Authority (EVIRA), 00790 Helsinki, Finland. (8) Cancer Gene Therapy Group, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland; Department of Obstetrics and Gynecology, Helsinki University Hospital, 00290 Helsinki, Finland. (9) TILT Biotherapeutics Ltd., 00290 Helsinki, Finland; Cancer Gene Therapy Group, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland; Helsinki University Hospital Comprehensive Cancer Center, 00290 Helsinki, Finland. Electronic address: akseli.hemminki@helsinki.fi.

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Influence of retronectin-mediated T cell activation on expansion and phenotype of CD19-specific chimeric antigen receptor T cells

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Introduction Enhanced in vivo expansion, long-term persistence of chimeric antigen receptor T (CART) cells and efficient tumor eradication through these cells are linked to the proportion of less-differentiated cells in the CART cell product. Retronectin is well established as an adjuvant for improved retroviral transduction while its property to enrich less-differentiated T cells is less known. In order to increase these subsets, we investigated effects of retronectin-mediated T cell activation for CD19-specific CART cell production. Materials and Methods Peripheral blood mononuclear cells (PBMCs) of healthy donors (HDs) and untreated chronic lymphocytic leukemia (CLL) patients without or with positive selection for CD3+ T cells were transduced with a CD19.CAR.CD28.CD137zeta 3rd generation retroviral vector. Activation of PBMCs was performed by CD3/CD28, CD3/CD28/retronectin or CD3/retronectin. Interleukin (IL)-7 and IL-15 were supplemented to all cultures. Retronectin was used in all three activation protocols for retroviral transduction. Expansion was assessed by trypan blue staining. Viability, transduction efficiency, immune phenotype and cytokine production were longitudinally analyzed by flow cytometry. Cytotoxic capacity of generated CART cells was evaluated using a classical chromium-51 release assay. Results Retronectin-mediated activation resulted in an enrichment of CD8+ cytotoxic CART cells and less-differentiated naive-like T cells (CD45RA+CCR7+). Retronectin-activated CART cells showed increased cytotoxic activity. However, activation with retronectin decreased viability, expansion, transduction efficiency and cytokine production, particularly of CLL patient-derived CART cells. Conclusion Both retronectin-mediated activation protocols promoted a less-differentiated CART cell phenotype without comprising cytotoxic properties of HD-derived CART cells. However, up-front retronectin resulted in reduced viability and expansion in CLL patients. This effect is probably attributed to the retronectin-mediated activation of B cells with prolonged CLL-persistence. Consequently, CART cell expansion and generation failed. In summary, activation with retronectin should be performed with caution and may be limited to patients without a higher percentage of tumor cells in the peripheral blood.

Author Info: (1) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany ; sophia.stock@med.uni-heidelberg.de. (2) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany

Author Info: (1) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany ; sophia.stock@med.uni-heidelberg.de. (2) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany ; Jean-Marc.Hoffmann@med.uni-heidelberg.de. (3) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany ; Maria-Luisa.Schubert@med.uni-heidelberg.de. (4) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany ; Wang.Lei@med.uni-heidelberg.de. (5) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany ; sanmei.wang@med.uni-heidelberg.de. (6) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany ; Wenjie.Gong@med.uni-heidelberg.de. (7) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany ; Brigitte.Neuber@med.uni-heidelberg.de. (8) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany ; Ulrike.Gern@med.uni-heidelberg.de. (9) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany ; Anita.Schmitt@med.uni-heidelberg.de. (10) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany. National Center for Tumor Diseases (NCT), German Cancer Consortium (DKTK) , Heidelberg, Germany ; Carsten.Mueller-Tidow@med.uni-heidelberg.de. (11) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany. National Center for Tumor Diseases (NCT), German Cancer Consortium (DKTK) , Heidelberg, Germany ; Peter.Dreger@med.uni-heidelberg.de. (12) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany. National Center for Tumor Diseases (NCT), German Cancer Consortium (DKTK) , Heidelberg, Germany ; Michael.Schmitt@med.uni-heidelberg.de. (13) Heidelberg University Hospital, Department of Internal Medicine V, Heidelberg, Germany. National Center for Tumor Diseases (NCT), German Cancer Consortium (DKTK) , Heidelberg, Germany ; Leopold.Sellner@med.uni-heidelberg.de.

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