Journal Articles

Innovative Methods

Methods with focus on improving cancer immunotherapy approaches

The route of administration dictates the immunogenicity of peptide-based cancer vaccines in mice

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Vaccines consisting of synthetic peptides representing cytotoxic T-lymphocyte (CTL) epitopes have long been considered as a simple and cost-effective approach to treat cancer. However, the efficacy of these vaccines in the clinic in patients with measurable disease remains questionable. We believe that the poor performance of peptide vaccines is due to their inability to generate sufficiently large CTL responses that are required to have a positive impact against established tumors. Peptide vaccines to elicit CTLs in the clinic have routinely been administered in the same manner as vaccines designed to induce antibody responses: injected subcutaneously and in many instances using Freund's adjuvant. We report here that peptide vaccines and poly-ICLC adjuvant administered via the unconventional intravenous route of immunization generate substantially higher CTL responses as compared to conventional subcutaneous injections, resulting in more successful antitumor effects in mice. Furthermore, amphiphilic antigen constructs such as palmitoylated peptides were shown to be better immunogens than long peptide constructs, which now are in vogue in the clinic. The present findings if translated into the clinical setting could help dissipate the wide-spread skepticism of whether peptide vaccines will ever work to treat cancer.

Author Info: (1) Cancer Immunology, Inflammation and Tolerance Program, Georgia Cancer Center, Augusta University, 1410 Laney Walker Blvd., CN-4142, Augusta, GA, 30912, USA. Washington University School of

Author Info: (1) Cancer Immunology, Inflammation and Tolerance Program, Georgia Cancer Center, Augusta University, 1410 Laney Walker Blvd., CN-4142, Augusta, GA, 30912, USA. Washington University School of Medicine, Saint Louis, MO, USA. (2) Cancer Immunology, Inflammation and Tolerance Program, Georgia Cancer Center, Augusta University, 1410 Laney Walker Blvd., CN-4142, Augusta, GA, 30912, USA. Department of Otolaryngology-Head and Neck Surgery, Asahikawa Medical University, Asahikawa, Japan. Department of Innovative Head and Neck Cancer Research and Treatment (IHNCRT), Asahikawa Medical University, Asahikawa, Japan. (3) Department of Otolaryngology-Head and Neck Surgery, Asahikawa Medical University, Asahikawa, Japan. Department of Pathology, Asahikawa Medical University, Asahikawa, Japan. (4) Cancer Immunology, Inflammation and Tolerance Program, Georgia Cancer Center, Augusta University, 1410 Laney Walker Blvd., CN-4142, Augusta, GA, 30912, USA. (5) Oncovir, Inc., Washington, DC, USA. (6) Cancer Immunology, Inflammation and Tolerance Program, Georgia Cancer Center, Augusta University, 1410 Laney Walker Blvd., CN-4142, Augusta, GA, 30912, USA. ecelis@augusta.edu.

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Natural modulators of the hallmarks of immunogenic cell death

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Natural compounds act as immunoadjuvants as their therapeutic effects trigger cancer stress response and release of damage-associated molecular patterns (DAMPs). These reactions occur through an increase in the immunogenicity of cancer cells that undergo stress followed by immunogenic cell death (ICD). These processes result in a chemotherapeutic response with a potent immune-mediating reaction. Natural compounds that induce ICD may function as an interesting approach in converting cancer into its own vaccine. However, multiple parameters determine whether a compound can act as an ICD inducer, including the nature of the inducer, the premortem stress pathways, the cell death pathways, the intrinsic antigenicity of the cell, and the potency and availability of an immune cell response. Thus, the identification of hallmarks of ICD is important in determining the prognostic biomarkers for new therapeutic approaches and combination treatments.

Author Info: (1) Laboratoire de Biologie Moleculaire et Cellulaire du Cancer, Hopital Kirchberg 9, rue Edward Steichen, L-2540 Luxembourg, Luxembourg. (2) Laboratoire de Biologie Moleculaire et Cellulaire

Author Info: (1) Laboratoire de Biologie Moleculaire et Cellulaire du Cancer, Hopital Kirchberg 9, rue Edward Steichen, L-2540 Luxembourg, Luxembourg. (2) Laboratoire de Biologie Moleculaire et Cellulaire du Cancer, Hopital Kirchberg 9, rue Edward Steichen, L-2540 Luxembourg, Luxembourg. (3) College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea. Electronic address: marcdiederich@snu.ac.kr.

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Follicular regulatory T cells infiltrated the ovarian carcinoma and resulted in CD8 T cell dysfunction dependent on IL-10 pathway

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A high Treg/CD8 T cell ratio in ovarian carcinoma was negatively associated with the prognosis of the patients. The human follicular regulatory T (Tfr) cells are a newly characterized subset of Treg cells with features of both follicular helper T (Tfh) cells (CXCR5(+)) and canonical Treg cells (CD25(+)Foxp3(+)). The role of Tfr cells in ovarian cancer is yet unclear. We found that in peripheral blood, the ovarian cancer patients presented significantly higher levels of both CD4(+)CD25(+)CD127(-)CXCR5(+) T cells and CD4(+)CD25(+)CD127(-)CXCR5(+)Foxp3(+) T cells than the healthy controls. In resected tumor samples, Tfr cells represented a much greater percentage of lymphocytes than in peripheral blood. Interestingly, the circulating Tfr cells from ovarian cancer patients presented significantly higher TGFB1 and IL10 expression than their counterparts in healthy controls directly ex vivo, and significantly higher IL10 after stimulation. The tumor-infiltrating Tfr cells presented further upregulated expression of TGFB1 and IL10. In addition, the levels of TGFB1 and IL10 expression by Tfr cells negatively associated with the expression of IFNG in tumor-infiltrating CD8 T cells. In an in vitro CD8 T cell/Tfr cell coculture system, we found that Tfr cells could significantly suppress the activation of CD8 T cells, in a manner that was dependent on IL-10 and probably on TGF-beta. Overall, our study found that Tfr cells could suppress CD8 T cells, and in ovarian cancer patients, the Tfr cells were increased in both frequency and function.

Author Info: (1) Department of Gynecology, Third Affiliated Hospital, Xinjiang Medical University, Urumqi 830011, China. Electronic address: lili_ulmq@sina.com. (2) Department of Gynecology, Third Affiliated Hospital, Xinjiang Medical

Author Info: (1) Department of Gynecology, Third Affiliated Hospital, Xinjiang Medical University, Urumqi 830011, China. Electronic address: lili_ulmq@sina.com. (2) Department of Gynecology, Third Affiliated Hospital, Xinjiang Medical University, Urumqi 830011, China. (3) Department of Gynecology, Third Affiliated Hospital, Xinjiang Medical University, Urumqi 830011, China.

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Anti-tumor macrophages activated by ferumoxytol combined or surface-functionalized with the TLR3 agonist poly (I : C) promote melanoma regression

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Macrophages orchestrate inflammation and control the promotion or inhibition of tumors and metastasis. Ferumoxytol (FMT), a clinically approved iron oxide nanoparticle, possesses anti-tumor therapeutic potential by inducing pro-inflammatory macrophage polarization. Toll-like receptor 3 (TLR3) activation also potently enhances the anti-tumor response of immune cells. Herein, the anti-tumor potential of macrophages harnessed by FMT combined with the TLR3 agonist, poly (I:C) (PIC), and FP-NPs (nanoparticles composed of amino-modified FMT (FMT-NH2) surface functionalized with PIC) was explored. Methods: Proliferation of B16F10 cells co-cultured with macrophages was measured using immunofluorescence or flow cytometry (FCM). Phagocytosis was analyzed using FCM and fluorescence imaging. FP-NPs were prepared through electrostatic interactions and their properties were characterized using dynamic light scattering, transmission electron microscopy, and gel retardation assay. Anti-tumor and anti-metastasis effects were evaluated in B16F10 tumor-bearing mice, and tumor-infiltrating immunocytes were detected by immunofluorescence staining and FCM. Results: FMT, PIC, or the combination of both hardly impaired B16F10 cell viability. However, FMT combined with PIC synergistically inhibited their proliferation by shifting macrophages to a tumoricidal phenotype with upregulated TNF-alpha and iNOS, increased NO secretion and augmented phagocytosis induced by NOX2-derived ROS in vitro. Combined treatment with FMT/PIC and FMT-NH2/PIC respectively resulted in primary melanoma regression and alleviated pulmonary metastasis with elevated pro-inflammatory macrophage infiltration and upregulation of pro-inflammatory genes in vivo. In comparison, FP-NPs with properties of internalization by macrophages and accumulation in the lung produced a more pronounced anti-metastatic effect accompanied with decreased myeloid-derived suppressor cells, and tumor-associated macrophages shifted to M1 phenotype. In vitro mechanistic studies revealed that FP-NPs nanoparticles barely affected B16F10 cell viability, but specifically retarded their growth by steering macrophages to M1 phenotype through NF-kappaB signaling. Conclusion: FMT synergized with the TLR3 agonist PIC either in combination or as a nano-composition to induce macrophage activation for primary and metastatic melanoma regression, and the nano-composition of FP-NPs exhibited a more superior anti-metastatic efficacy.

Author Info: (1) The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing 210093, PR China. (2) MOE Key Laboratory of High

Author Info: (1) The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing 210093, PR China. (2) MOE Key Laboratory of High Performance Polymer Materials and Technology, Department of Polymer Science & Engineering, College of Chemistry & Chemical Engineering, and Jiangsu Key Laboratory for Nanotechnology, Nanjing University , Nanjing, 210093, PR China. (3) The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing 210093, PR China. (4) Department of Oncology, First Affiliated Hospital, Nanjing Medical University, Nanjing 211166, PR China. (5) Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing National Laboratory of Microstructures, Nanjing University, Nanjing, Jiangsu 210093, PR China. (6) The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing 210093, PR China. (7) General Clinical Research Center, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, PR China. (8) The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing 210093, PR China. Jiangsu Key Laboratory of Molecular Medicine, Nanjing University, Nanjing 210093, PR China.

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T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

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Cancer immunotherapy has proven high efficacy in treating diverse cancer entities by immune checkpoint modulation and adoptive T-cell transfer. However, patterns of treatment response differ substantially from conventional therapies, and reliable surrogate markers are missing for early detection of responders versus non-responders. Current imaging techniques using (18)F-fluorodeoxyglucose-positron-emmission-tomograpy ((18)F-FDG-PET) cannot discriminate, at early treatment times, between tumor progression and inflammation. Therefore, direct imaging of T cells at the tumor site represents a highly attractive tool to evaluate effective tumor rejection or evasion. Moreover, such markers may be suitable for theranostic imaging. Methods: We mainly investigated the potential of two novel pan T-cell markers, CD2 and CD7, for T-cell tracking by immuno-PET imaging. Respective antibody- and F(ab )2 fragment-based tracers were produced and characterized, focusing on functional in vitro and in vivo T-cell analyses to exclude any impact of T-cell targeting on cell survival and antitumor efficacy. Results: T cells incubated with anti-CD2 and anti-CD7 F(ab )2 showed no major modulation of functionality in vitro, and PET imaging provided a distinct and strong signal at the tumor site using the respective zirconium-89-labeled radiotracers. However, while T-cell tracking by anti-CD7 F(ab )2 had no long-term impact on T-cell functionality in vivo, anti-CD2 F(ab )2 caused severe T-cell depletion and failure of tumor rejection. Conclusion: This study stresses the importance of extended functional T-cell assays for T-cell tracer development in cancer immunotherapy imaging and proposes CD7 as a highly suitable target for T-cell immuno-PET imaging.

Author Info: (1) Clinic and Policlinic for Internal Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (2) Clinic and Policlinic for Internal Medicine

Author Info: (1) Clinic and Policlinic for Internal Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (2) Clinic and Policlinic for Internal Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. German Cancer Consortium (DKTK), partner-site Munich; and German Cancer Research Center (DKFZ), Heidelberg, Germany. (3) Department of Nuclear Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (4) Clinic and Policlinic for Internal Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (5) German Cancer Consortium (DKTK), partner-site Munich; and German Cancer Research Center (DKFZ), Heidelberg, Germany. Institute of Pathology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (6) Department of Nuclear Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (7) Clinic and Policlinic for Internal Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (8) Clinic and Policlinic for Internal Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (9) Clinic and Policlinic for Internal Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (10) Clinic and Policlinic for Internal Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (11) Clinic and Policlinic for Internal Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (12) Clinic and Policlinic for Internal Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. German Cancer Consortium (DKTK), partner-site Munich; and German Cancer Research Center (DKFZ), Heidelberg, Germany. (13) German Cancer Consortium (DKTK), partner-site Munich; and German Cancer Research Center (DKFZ), Heidelberg, Germany. Institute of Pathology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (14) German Cancer Consortium (DKTK), partner-site Munich; and German Cancer Research Center (DKFZ), Heidelberg, Germany. Department of Nuclear Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (15) German Cancer Consortium (DKTK), partner-site Munich; and German Cancer Research Center (DKFZ), Heidelberg, Germany. Department of Nuclear Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (16) Department of Nuclear Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. (17) Clinic and Policlinic for Internal Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany. German Cancer Consortium (DKTK), partner-site Munich; and German Cancer Research Center (DKFZ), Heidelberg, Germany.

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Arg1 expression defines immunosuppressive subsets of tumor-associated macrophages

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Tumor-associated macrophages (TAM) have attracted attention as they can modulate key cancer-related activities, yet TAM represent a heterogenous group of cells that remain incompletely characterized. In growing tumors, TAM are often referred to as M2-like macrophages, which are cells that display immunosuppressive and tumorigenic functions and express the enzyme arginase 1 (Arg1). Methods: Here we combined high resolution intravital imaging with single cell RNA seq to uncover the topography and molecular profiles of immunosuppressive macrophages in mice. We further assessed how immunotherapeutic interventions impact these cells directly in vivo. Results: We show that: i) Arg1+ macrophages are more abundant in tumors compared to other organs; ii) there exist two morphologically distinct subsets of Arg1 TAM defined by previously unknown markers (Gbp2b, Bst1, Sgk1, Pmepa1, Ms4a7); iii) anti-Programmed Cell Death-1 (aPD-1) therapy decreases the number of Arg1+ TAM while increasing Arg1- TAM; iv) accordingly, pharmacological inhibition of arginase 1 does not synergize with aPD-1 therapy. Conclusion: Overall, this research shows how powerful complementary single cell analytical approaches can be used to improve our understanding of drug action in vivo.

Author Info: (1) Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA 02114. (2) Center for Systems Biology, Massachusetts General Hospital, 18

Author Info: (1) Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA 02114. (2) Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA 02114. (3) Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA 02114. (4) Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA 02114. (5) Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA 02114. (6) Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA 02114. (7) Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA 02114. Department of Systems Biology, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115.

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Delivery technologies for cancer immunotherapy

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Immunotherapy has become a powerful clinical strategy for treating cancer. The number of immunotherapy drug approvals has been increasing, with numerous treatments in clinical and preclinical development. However, a key challenge in the broad implementation of immunotherapies for cancer remains the controlled modulation of the immune system, as these therapeutics have serious adverse effects including autoimmunity and nonspecific inflammation. Understanding how to increase the response rates to various classes of immunotherapy is key to improving efficacy and controlling these adverse effects. Advanced biomaterials and drug delivery systems, such as nanoparticles and the use of T cells to deliver therapies, could effectively harness immunotherapies and improve their potency while reducing toxic side effects. Here, we discuss these research advances, as well as the opportunities and challenges for integrating delivery technologies into cancer immunotherapy, and we critically analyse the outlook for these emerging areas.

Author Info: (1) Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA. (2) Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA, USA. Abramson Cancer Center, Perelman

Author Info: (1) Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA. (2) Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA, USA. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. (3) Department of Chemical Engineering and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. rlanger@mit.edu. (4) Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA. mjmitch@seas.upenn.edu. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. mjmitch@seas.upenn.edu.

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Visualizing Interactions of Circulating Tumor Cell and Dendritic Cell in the Blood Circulation Using In Vivo Imaging Flow Cytometry

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OBJECTIVE: Visualizing cell interactions in blood circulation is of great importance in studies of anticancer immunotherapy or drugs. However, the lack of a suitable imaging system hampers progress in this field. METHODS: In this work, we built a dual-channel in vivo imaging flow cytometer to visualize the interactions of circulating tumor cells (CTCs) and dendritic cells (DCs) simultaneously in the bloodstream. Two artificial neural networks were trained to identify blood vessels and cells in the acquired images. RESULTS AND CONCLUSION: Using this technique, single CTCs and CTC clusters were readily distinguished by their morphology. Interactions of CTCs and DCs were identified, while their moving velocities were analyzed. The CTC-DC clusters moved at a slower velocity than that of single CTCs or DCs. This may provide new insights into tumor metastasis and blood rheology. SIGNIFICANCE: This in vivo imaging flow cytometry system holds great potential for assessing the efficiency of targeting CTCs with anticancer immune cells or drugs.

Author Info: (1) (2) (3) (4) (5) (6) (7) (8)

Author Info: (1) (2) (3) (4) (5) (6) (7) (8)

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Modularly engineered injectable hybrid hydrogels based on protein-polymer network as potent immunologic adjuvant in vivo

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Lymphoid organs, which are populated by dendritic cells (DCs), are highly specialized tissues and provide an ideal microenvironment for T-cell priming. However, intramuscular or subcutaneous delivery of vaccine to DCs, a subset of antigen-presenting cells, has failed to stimulate optimal immune response for effective vaccination and need for adjuvants to induce immune response. To address this issue, we developed an in situ-forming injectable hybrid hydrogel that spontaneously assemble into microporous network upon subcutaneous administration, which provide a cellular niche to host immune cells, including DCs. In situ-forming injectable hybrid hydrogelators, composed of protein-polymer conjugates, formed a hydrogel depot at the close proximity to the dermis, resulting in a rapid migration of immune cells to the hydrogel boundary and infiltration to the microporous network. The biocompatibility of the watery microporous network allows recruitment of DCs without a DC enhancement factor, which was significantly higher than that of traditional hydrogel releasing chemoattractants, granulocyte-macrophage colony-stimulating factor. Owing to the sustained degradation of microporous hydrogel network, DNA vaccine release can be sustained, and the recruitment of DCs and their homing to lymph node can be modulated. Furthermore, immunization of a vaccine encoding amyloid-beta fusion proteinbearing microporous network induced a robust antigen-specific immune response in vivo and strong recall immune response was exhibited due to immunogenic memory. These hybrid hydrogels can be administered in a minimally invasive manner using hypodermic needle, bypassing the need for cytokine or DC enhancement factor and provide niche to host immune cells. These findings highlight the potential of hybrid hydrogels that may serve as a simple, yet multifunctional, platform for DNA vaccine delivery to modulate immune response.

Author Info: (1) Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, Vietnam. (2) School of Chemical Engineering, Theranostic Macromolecules Research Center, Sungkyunkwan University

Author Info: (1) Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, Vietnam. (2) School of Chemical Engineering, Theranostic Macromolecules Research Center, Sungkyunkwan University, Suwon 16419, Republic of Korea. (3) School of Chemical Engineering, Theranostic Macromolecules Research Center, Sungkyunkwan University, Suwon 16419, Republic of Korea. (4) School of Chemical Engineering, Theranostic Macromolecules Research Center, Sungkyunkwan University, Suwon 16419, Republic of Korea. (5) School of Chemical Engineering, Theranostic Macromolecules Research Center, Sungkyunkwan University, Suwon 16419, Republic of Korea. (6) School of Pharmacy, Theranostic Macromolecules Research Center, Sungkyunkwan University, Suwon 16419, Republic of Korea. (7) School of Chemical Engineering, Theranostic Macromolecules Research Center, Sungkyunkwan University, Suwon 16419, Republic of Korea. (8) School of Chemical Engineering, Theranostic Macromolecules Research Center, Sungkyunkwan University, Suwon 16419, Republic of Korea. (9) School of Pharmacy, Theranostic Macromolecules Research Center, Sungkyunkwan University, Suwon 16419, Republic of Korea. Electronic address: jhjeong@skku.edu. (10) School of Chemical Engineering, Theranostic Macromolecules Research Center, Sungkyunkwan University, Suwon 16419, Republic of Korea. Electronic address: dslee@skku.edu.

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Development of a Novel Anti-CD19 Chimeric Antigen Receptor: A Paradigm for an Affordable CAR T Cell Production at Academic Institutions

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Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells in vitro, inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation. In vivo, A3B1 CAR T cells are able to fully control disease progression in an NOD.Cg-Prkdc (scid) Il2rd (tm1Wjl) /SzJ (NSG) xenograph B-ALL mouse model. Based on the pre-clinical data, we conclude that our CART19 is clearly functional against CD19+ cells, to a level similar to other CAR19s currently being used in the clinic. Concurrently, we describe the implementation of our CAR T cell production system, using lentiviral vector and CliniMACS Prodigy, within a medium-sized academic institution. The results of the validation phase show our system is robust and reproducible, while maintaining a low cost that is affordable for academic institutions. Our model can serve as a paradigm for similar institutions, and it may help to make CAR T cell treatment available to all patients.

Author Info: (1) Department of Hematology, ICMHO, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153

Author Info: (1) Department of Hematology, ICMHO, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. (2) Department of Immunology, CDB, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. (3) Stem Cells and Regenerative Medicine Laboratory, Production and Validation Center of Advanced Therapies (Creatio), Department of Biomedical Sciences, University of Barcelona, Casanova 143, 08036 Barcelona, Spain. (4) Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. Physiopathology and Molecular Bases in Hematology Group, IDIBAPS, Rossello 153, 08036 Barcelona, Spain. (5) Department of Hematology, ICMHO, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. (6) Department of Immunology, CDB, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Institut de Recerca Pediatrica Hospital Sant Joan de Deu, Universidad de Barcelona, Passeig de Sant Joan de Deu, 2, 08950 Esplugues de Llobregat, Barcelona, Spain. (7) Department of Immunology, CDB, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. (8) Department of Hematology, ICMHO, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. (9) Department of Hematology, ICMHO, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. (10) Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Casanova 143, 08036 Barcelona, Spain. (11) Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Casanova 143, 08036 Barcelona, Spain. (12) Department of Pathology, Hospital Clinic, IDIBAPS, Villarroel 170, 08036 Barcelona, Spain. (13) Department of Immunology, CDB, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. (14) Department of Immunology, CDB, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Universitat de Barcelona, Casanova 143, 08036 Barcelona, Spain. (15) Department of Immunology, CDB, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. Immunology Unit, Department of Biomedical Sciences, University of Barcelona, Casanova 143, 08036 Barcelona, Spain. (16) Department of Immunology, CDB, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. (17) Department of Immunology, CDB, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. (18) Department of Hematology, ICMHO, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. (19) Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. Department of Hemotherapy and Hemostasis, ICMHO, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. (20) Unit of Advanced Therapies, Hospital Clinic de Barcelona, Blood and Tissue Bank -BST-, Passeig del Taulat, 106, 08005 Barcelona, Spain. (21) Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. Stem Cells and Regenerative Medicine Laboratory, Production and Validation Center of Advanced Therapies (Creatio), Department of Biomedical Sciences, University of Barcelona, Casanova 143, 08036 Barcelona, Spain. Universitat de Barcelona, Casanova 143, 08036 Barcelona, Spain. (22) Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. Department of Hemotherapy and Hemostasis, ICMHO, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. (23) Department of Hematology, ICMHO, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. (24) Unit of Advanced Therapies, Hospital Clinic de Barcelona, Blood and Tissue Bank -BST-, Passeig del Taulat, 106, 08005 Barcelona, Spain. (25) Unit of Advanced Therapies, Hospital Clinic de Barcelona, Blood and Tissue Bank -BST-, Passeig del Taulat, 106, 08005 Barcelona, Spain. (26) Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. Physiopathology and Molecular Bases in Hematology Group, IDIBAPS, Rossello 153, 08036 Barcelona, Spain. Department of Pathology, Hospital Clinic, IDIBAPS, Villarroel 170, 08036 Barcelona, Spain. Universitat de Barcelona, Casanova 143, 08036 Barcelona, Spain. Centro de Investigacion Biomedica en Red de Cancer (ISCIII-CIBERONC), Barcelona, Spain. (27) Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Casanova 143, 08036 Barcelona, Spain. Centro de Investigacion Biomedica en Red de Cancer (ISCIII-CIBERONC), Barcelona, Spain. Institucio Catalana de Recerca i Estudis Avancats (ICREA), Barcelona, Spain. (28) Department of Hematology, ICMHO, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Casanova 143, 08036 Barcelona, Spain. Universitat de Barcelona, Casanova 143, 08036 Barcelona, Spain. (29) Department of Immunology, CDB, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. Universitat de Barcelona, Casanova 143, 08036 Barcelona, Spain. (30) Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. Physiopathology and Molecular Bases in Hematology Group, IDIBAPS, Rossello 153, 08036 Barcelona, Spain. Centro de Investigacion Biomedica en Red de Cancer (ISCIII-CIBERONC), Barcelona, Spain. (31) Institut de Recerca Pediatrica Hospital Sant Joan de Deu, Universidad de Barcelona, Passeig de Sant Joan de Deu, 2, 08950 Esplugues de Llobregat, Barcelona, Spain. (32) Department of Hematology, ICMHO, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. Universitat de Barcelona, Casanova 143, 08036 Barcelona, Spain. Centro de Investigacion Biomedica en Red de Cancer (ISCIII-CIBERONC), Barcelona, Spain. (33) Department of Immunology, CDB, Hospital Clinic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. Institut d'Investigacions Biomediques August Pi i Sunyer - IDIBAPS, Rossello 153, 08036 Barcelona, Spain. Institut de Recerca Pediatrica Hospital Sant Joan de Deu, Universidad de Barcelona, Passeig de Sant Joan de Deu, 2, 08950 Esplugues de Llobregat, Barcelona, Spain. Universitat de Barcelona, Casanova 143, 08036 Barcelona, Spain.

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