Journal Articles

Chimeric Antigen Receptor Designed to Prevent Ubiquitination and Downregulation Showed Durable Antitumor Efficacy

ABSTRACT: Clinical evidence suggests that poor persistence of chimeric antigen receptor-T cells (CAR-T) in patients limits therapeutic efficacy. Here, we designed a CAR with recyclable capability to promote in vivo persistence and to sustain antitumor activity. We showed that the engagement of tumor antigens induced rapid ubiquitination of CARs, causing CAR downmodulation followed by lysosomal degradation. Blocking CAR ubiquitination by mutating all lysines in the CAR cytoplasmic domain (CARKR) markedly repressed CAR downmodulation by inhibiting lysosomal degradation while enhancing recycling of internalized CARs back to the cell surface. Upon encountering tumor antigens, CARKR-T cells ameliorated the loss of surface CARs, which promoted their long-term killing capacity. Moreover, CARKR-T cells containing 4-1BB signaling domains displayed elevated endosomal 4-1BB signaling that enhanced oxidative phosphorylation and promoted memory T cell differentiation, leading to superior persistence in vivo. Collectively, our study provides a straightforward strategy to optimize CAR-T antitumor efficacy by redirecting CAR trafficking.

Author Info: (1) School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell S

Author Info: (1) School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China; University of Chinese Academy of Sciences, Beijing 100049, China. (2) School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. (3) ENT institute and Department of Otorhinolaryngology, Eye & ENT Hospital, Fudan University, Shanghai 200031, China. (4) State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China. (5) Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China. (6) School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China. (7) Center for Quantitative Biology and Peking-Tsinghua Joint Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China. (8) ENT institute and Department of Otorhinolaryngology, Eye & ENT Hospital, Fudan University, Shanghai 200031, China. Electronic address: eentwuhaitao@163.com. (9) State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China; School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China. Electronic address: cqxu@sibcb.ac.cn. (10) School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address: wanghp@shanghaitech.edu.cn.

Type 1 conventional dendritic cells are systemically dysregulated early in pancreatic carcinogenesis

ABSTRACT: Type 1 conventional dendritic cells (cDC1s) are typically thought to be dysregulated secondarily to invasive cancer. Here, we report that cDC1 dysfunction instead develops in the earliest stages of preinvasive pancreatic intraepithelial neoplasia (PanIN) in the KrasLSL-G12D/+ Trp53LSL-R172H/+ Pdx1-Cre-driven (KPC) mouse model of pancreatic cancer. cDC1 dysfunction is systemic and progressive, driven by increased apoptosis, and results in suboptimal up-regulation of T cell-polarizing cytokines during cDC1 maturation. The underlying mechanism is linked to elevated IL-6 concomitant with neoplasia. Neutralization of IL-6 in vivo ameliorates cDC1 apoptosis, rescuing cDC1 abundance in tumor-bearing mice. CD8+ T cell response to vaccination is impaired as a result of cDC1 dysregulation. Yet, combination therapy with CD40 agonist and Flt3 ligand restores cDC1 abundance to normal levels, decreases cDC1 apoptosis, and repairs cDC1 maturation to drive superior control of tumor outgrowth. Our study therefore reveals the unexpectedly early and systemic onset of cDC1 dysregulation during pancreatic carcinogenesis and suggests therapeutically tractable strategies toward cDC1 repair.

Author Info: (1) Immunology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA. (2) Department of Medicine, Perelman School of Medicine, University of Pen

Author Info: (1) Immunology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA. (2) Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA. (3) Cell and Molecular Biology Graduate Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA. (4) Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA. (5) Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA. (6) Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

Necroptosis in Immuno-Oncology and Cancer Immunotherapy

Immune-checkpoint blockers (ICBs) have revolutionized oncology and firmly established the subfield of immuno-oncology. Despite this renaissance, a subset of cancer patients remain unresponsive to ICBs due to widespread immuno-resistance. To "break" cancer cell-driven immuno-resistance, researchers have long floated the idea of therapeutically facilitating the immunogenicity of cancer cells by disrupting tumor-associated immuno-tolerance via conventional anticancer therapies. It is well appreciated that anticancer therapies causing immunogenic or inflammatory cell death are best positioned to productively activate anticancer immunity. A large proportion of studies have emphasized the importance of immunogenic apoptosis (i.e., immunogenic cell death or ICD); yet, it has also emerged that necroptosis, a programmed necrotic cell death pathway, can also be immunogenic. Emergence of a proficient immune profile for necroptosis has important implications for cancer because resistance to apoptosis is one of the major hallmarks of tumors. Putative immunogenic or inflammatory characteristics driven by necroptosis can be of great impact in immuno-oncology. However, as is typical for a highly complex and multi-factorial disease like cancer, a clear cause versus consensus relationship on the immunobiology of necroptosis in cancer cells has been tough to establish. In this review, we discuss the various aspects of necroptosis immunobiology with specific focus on immuno-oncology and cancer immunotherapy.

Author Info: (1) Department of Cellular and Molecular Medicine, Laboratory of Cell Stress & Immunity (CSI), KU Leuven, 3000 Leuven, Belgium. (2) Department of Cellular and Molecular Medicine, L

Author Info: (1) Department of Cellular and Molecular Medicine, Laboratory of Cell Stress & Immunity (CSI), KU Leuven, 3000 Leuven, Belgium. (2) Department of Cellular and Molecular Medicine, Laboratory of Cell Stress & Immunity (CSI), KU Leuven, 3000 Leuven, Belgium. (3) Department of Cellular and Molecular Medicine, Laboratory of Cell Stress & Immunity (CSI), KU Leuven, 3000 Leuven, Belgium. (4) Department of Cellular and Molecular Medicine, Laboratory of Cellular Transport Systems, KU Leuven, 3000 Leuven, Belgium. (5) Department of Cellular and Molecular Medicine, Laboratory of Cellular Transport Systems, KU Leuven, 3000 Leuven, Belgium. (6) Department of Microbiology, Immunology and Transplantation, KU Leuven, 3000 Leuven, Belgium. (7) Department of Human Structure and Repair, Cell Death Investigation and Therapy Laboratory, Ghent University, 9000 Ghent, Belgium. Department of Pathophysiology, Sechenov First Moscow State Medical University (Sechenov University), 119146 Moscow, Russia. (8) Department of Cellular and Molecular Medicine and Leuven Kanker Instituut (LKI), Laboratory of Molecular and Cellular Signaling, KU Leuven, 3000 Leuven, Belgium. (9) Department of Cellular and Molecular Medicine and Leuven Kanker Instituut (LKI), Laboratory of Molecular and Cellular Signaling, KU Leuven, 3000 Leuven, Belgium. (10) Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent, Belgium. VIB Center for Inflammation Research, 9052 Ghent, Belgium. Methusalem Program, Ghent University, 9000 Ghent, Belgium. (11) Department of Cellular and Molecular Medicine, Laboratory of Cell Stress & Immunity (CSI), KU Leuven, 3000 Leuven, Belgium.

Antibody-Drug Conjugates: The New Frontier of Chemotherapy

In recent years, antibody-drug conjugates (ADCs) have become promising antitumor agents to be used as one of the tools in personalized cancer medicine. ADCs are comprised of a drug with cytotoxic activity cross-linked to a monoclonal antibody, targeting antigens expressed at higher levels on tumor cells than on normal cells. By providing a selective targeting mechanism for cytotoxic drugs, ADCs improve the therapeutic index in clinical practice. In this review, the chemistry of ADC linker conjugation together with strategies adopted to improve antibody tolerability (by reducing antigenicity) are examined, with particular attention to ADCs approved by the regulatory agencies (the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA)) for treating cancer patients. Recent developments in engineering Immunoglobulin (Ig) genes and antibody humanization have greatly reduced some of the problems of the first generation of ADCs, beset by problems, such as random coupling of the payload and immunogenicity of the antibody. ADC development and clinical use is a fast, evolving area, and will likely prove an important modality for the treatment of cancer in the near future.

Author Info: (1) Department of Life, Health and Environmental Sciences, University of L'Aquila, I-67100 L'Aquila, Italy. (2) MediaPharma SrL, I-66013 Chieti, Italy. (3) Department of Life, Heal

Author Info: (1) Department of Life, Health and Environmental Sciences, University of L'Aquila, I-67100 L'Aquila, Italy. (2) MediaPharma SrL, I-66013 Chieti, Italy. (3) Department of Life, Health and Environmental Sciences, University of L'Aquila, I-67100 L'Aquila, Italy. (4) Department of Life, Health and Environmental Sciences, University of L'Aquila, I-67100 L'Aquila, Italy. (5) Department of Life, Health and Environmental Sciences, University of L'Aquila, I-67100 L'Aquila, Italy. (6) MediaPharma SrL, I-66013 Chieti, Italy. (7) MediaPharma SrL, I-66013 Chieti, Italy. (8) MediaPharma SrL, I-66013 Chieti, Italy. Department of Medical, Oral and Biotechnological Sciences, University of Chieti-Pescara, I-66100 Chieti, Italy. (9) Department of Medical, Oral and Biotechnological Sciences, University of Chieti-Pescara, I-66100 Chieti, Italy. (10) The Simon Flavell Leukaemia Research Laboratory, Southampton General Hospital, Southampton SO16 6YD, UK. (11) Department of Life, Health and Environmental Sciences, University of L'Aquila, I-67100 L'Aquila, Italy. (12) Department of Life, Health and Environmental Sciences, University of L'Aquila, I-67100 L'Aquila, Italy.

Natural Born Killers: NK Cells in Cancer Therapy

Cellular therapy has emerged as an attractive option for the treatment of cancer, and adoptive transfer of chimeric antigen receptor (CAR) expressing T cells has gained FDA approval in hematologic malignancy. However, limited efficacy was observed using CAR-T therapy in solid tumors. Natural killer (NK) cells are crucial for tumor surveillance and exhibit potent killing capacity of aberrant cells in an antigen-independent manner. Adoptive transfer of unmodified allogeneic or autologous NK cells has shown limited clinical benefit due to factors including low cell number, low cytotoxicity and failure to migrate to tumor sites. To address these problems, immortalized and autologous NK cells have been genetically engineered to express high affinity receptors (CD16), CARs directed against surface proteins (PD-L1, CD19, Her2, etc.) and endogenous cytokines (IL-2 and IL-15) that are crucial for NK cell survival and cytotoxicity, with positive outcomes reported by several groups both preclinically and clinically. With a multitude of NK cell-based therapies currently in clinic trials, it is likely they will play a crucial role in next-generation cell therapy-based treatment. In this review, we will highlight the recent advances and limitations of allogeneic, autologous and genetically enhanced NK cells used in adoptive cell therapy.

Author Info: (1) Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. (2) Laboratory of Tum

Author Info: (1) Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. (2) Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. (3) Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Characterization of Neoantigen Load Subgroups in Gynecologic and Breast Cancers

OBJECTIVE: Although gynecologic and breast (Pan-Gyn) cancers share a variety of similar characteristics, their response to immunotherapy is different. Immune checkpoint inhibitor therapy is not effective in all patients, while neoantigen load (NAL) may be a predictive biomarker. However, the selection of a NAL cutoff point and its predictive effect remain to be elucidated. METHODS: We divided 812 Pan-Gyn cancer samples from The Cancer Genome Atlas into three groups based on 60 and 80% of their load percentile. We then correlated the identified NAL subgroups with gene expression, somatic mutation, DNA methylation, and clinicopathological information. We also characterized each subgroup by distinct immune cell enrichment, PD-1 signaling, and cytolytic activity. Finally, we predicted the response of each subgroup to chemotherapy and immunotherapy. RESULTS: Across Pan-Gyn cancers, we identified three distinct NAL subgroups. These subgroups showed differences in biological function, genetic information, clinical variables, and immune infiltration. Eighty percent was identified as a meaningful cutoff point for NAL. In all patients, a higher NAL (top 20%) was associated with better overall survival as well as high immune infiltration and low intra-tumor heterogeneity. Furthermore, an interesting lncRNA named AC092580.4 was found, which was associated with two significantly different immune genes (CXCL9 and CXCL13). CONCLUSIONS: Our novel findings provide further insights into the NAL of Pan-Gyn cancers and may open up novel opportunities for their exploitation toward personalized treatment with immunotherapy.

Author Info: (1) State Key Laboratory of Natural Medicines, Research Center of Biostatistics and Computational Pharmacy, China Pharmaceutical University, Nanjing, China. (2) State Key Laborator

Author Info: (1) State Key Laboratory of Natural Medicines, Research Center of Biostatistics and Computational Pharmacy, China Pharmaceutical University, Nanjing, China. (2) State Key Laboratory of Natural Medicines, Research Center of Biostatistics and Computational Pharmacy, China Pharmaceutical University, Nanjing, China. (3) State Key Laboratory of Natural Medicines, Research Center of Biostatistics and Computational Pharmacy, China Pharmaceutical University, Nanjing, China. (4) State Key Laboratory of Natural Medicines, Research Center of Biostatistics and Computational Pharmacy, China Pharmaceutical University, Nanjing, China. (5) State Key Laboratory of Natural Medicines, Research Center of Biostatistics and Computational Pharmacy, China Pharmaceutical University, Nanjing, China. (6) State Key Laboratory of Natural Medicines, Research Center of Biostatistics and Computational Pharmacy, China Pharmaceutical University, Nanjing, China.

Identification of a peptide targeting CD56

Neural cell adhesion molecule 1 (NCAM1/CD56) is expressed on immune cells, myoblasts, and malignant cells, and there is a growing demand for the genetic detection of CD56 and CD56-targeted therapy. In the present study, we developed a novel peptide ligand (designated Natein) that binds to human CD56 by using T7 phage display technology. Natein recognized the extracellular region of CD56 and could bind to natural killer (NK) cells and CD56-positive (CD56(+)) cancer cells. CD56(+) cells enriched from human peripheral blood mononuclear cells (PBMCs) using biotinylated Natein-conjugated microbeads, similarly to CD56 antibody-isolated cells, demonstrated functional cytotoxicity against K562 cells. In addition, Natein could be used to stain CD56(+) lymphoma cells in nasal-type extranodal NK/T-cell lymphoma tissues similarly to a CD56 antibody. These findings suggest that Natein has the potential to be alternative to CD56 antibody that could be used for peptide-based cell isolation and diagnosis.

Author Info: (1) MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, PR China. (2) MOE Key Labora

Author Info: (1) MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, PR China. (2) MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, PR China. (3) The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, PR China. (4) Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, PR China. Electronic address: wanghua9@mail.sysu.edu.cn. (5) MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, PR China. Electronic address: zhang39@mail.sysu.edu.cn.

Dendritic cell biology and its role in tumor immunotherapy

As crucial antigen presenting cells, dendritic cells (DCs) play a vital role in tumor immunotherapy. Taking into account the many recent advances in DC biology, we discuss how DCs (1) recognize pathogenic antigens with pattern recognition receptors through specific phagocytosis and through non-specific micropinocytosis, (2) process antigens into small peptides with proper sizes and sequences, and (3) present MHC-peptides to CD4(+) and CD8(+) T cells to initiate immune responses against invading microbes and aberrant host cells. During anti-tumor immune responses, DC-derived exosomes were discovered to participate in antigen presentation. T cell microvillar dynamics and TCR conformational changes were demonstrated upon DC antigen presentation. Caspase-11-driven hyperactive DCs were recently reported to convert effectors into memory T cells. DCs were also reported to crosstalk with NK cells. Additionally, DCs are the most important sentinel cells for immune surveillance in the tumor microenvironment. Alongside DC biology, we review the latest developments for DC-based tumor immunotherapy in preclinical studies and clinical trials. Personalized DC vaccine-induced T cell immunity, which targets tumor-specific antigens, has been demonstrated to be a promising form of tumor immunotherapy in patients with melanoma. Importantly, allogeneic-IgG-loaded and HLA-restricted neoantigen DC vaccines were discovered to have robust anti-tumor effects in mice. Our comprehensive review of DC biology and its role in tumor immunotherapy aids in the understanding of DCs as the mentors of T cells and as novel tumor immunotherapy cells with immense potential.

Author Info: (1) State Key Laboratory of Respiratory Disease, Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, 510095, China. Laboratory of Oncology, Center fo

Author Info: (1) State Key Laboratory of Respiratory Disease, Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, 510095, China. Laboratory of Oncology, Center for Molecular Medicine, School of Basic Medicine, Faculty of Medicine, Yangtze University, 1 Nanhuan Road, Jingzhou, 434023, Hubei, China. Department of Biochemistry and Molecular Biology, School of Basic Medicine, Faculty of Medicine, Yangtze University, Jingzhou, 434023, Hubei, China. Department of Gynaecology, Comprehensive Cancer Center, Hannover Medical School, 30625, Hannover, Germany. (2) Laboratory of Oncology, Center for Molecular Medicine, School of Basic Medicine, Faculty of Medicine, Yangtze University, 1 Nanhuan Road, Jingzhou, 434023, Hubei, China. Department of Biochemistry and Molecular Biology, School of Basic Medicine, Faculty of Medicine, Yangtze University, Jingzhou, 434023, Hubei, China. (3) Stanford University, Stanford, CA, 94305, USA. (4) Laboratory of Oncology, Center for Molecular Medicine, School of Basic Medicine, Faculty of Medicine, Yangtze University, 1 Nanhuan Road, Jingzhou, 434023, Hubei, China. Department of Laboratory Medicine, School of Basic Medicine, Faculty of Medicine, Yangtze University, 1 Nanhuan Road, Jingzhou, 434023, Hubei, China. (5) Laboratory of Oncology, Center for Molecular Medicine, School of Basic Medicine, Faculty of Medicine, Yangtze University, 1 Nanhuan Road, Jingzhou, 434023, Hubei, China. Department of Pathophysiology, School of Basic Medicine, Faculty of Medicine, Yangtze University, Jingzhou, 434023, Hubei, China. (6) Laboratory of Oncology, Center for Molecular Medicine, School of Basic Medicine, Faculty of Medicine, Yangtze University, 1 Nanhuan Road, Jingzhou, 434023, Hubei, China. Department of Biochemistry and Molecular Biology, School of Basic Medicine, Faculty of Medicine, Yangtze University, Jingzhou, 434023, Hubei, China. Department of Medical Imaging, School of Basic Medicine, Faculty of Medicine, Yangtze University, Jingzhou, 434023, Hubei, China. (7) Laboratory of Oncology, Center for Molecular Medicine, School of Basic Medicine, Faculty of Medicine, Yangtze University, 1 Nanhuan Road, Jingzhou, 434023, Hubei, China. Department of Biochemistry and Molecular Biology, School of Basic Medicine, Faculty of Medicine, Yangtze University, Jingzhou, 434023, Hubei, China. (8) Department of Oncology, First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China. (9) Laboratory of Oncology, Center for Molecular Medicine, School of Basic Medicine, Faculty of Medicine, Yangtze University, 1 Nanhuan Road, Jingzhou, 434023, Hubei, China. Department of Biochemistry and Molecular Biology, School of Basic Medicine, Faculty of Medicine, Yangtze University, Jingzhou, 434023, Hubei, China. (10) Institute for Infectious Diseases and Endemic Diseases Prevention and Control, Beijing Center for Diseases Prevention and Control, Beijing, 100013, China. (11) State Key Laboratory of Respiratory Disease, Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, 510095, China. (12) Laboratory of Oncology, Center for Molecular Medicine, School of Basic Medicine, Faculty of Medicine, Yangtze University, 1 Nanhuan Road, Jingzhou, 434023, Hubei, China. zhaowu823@126.com. Department of Biochemistry and Molecular Biology, School of Basic Medicine, Faculty of Medicine, Yangtze University, Jingzhou, 434023, Hubei, China. zhaowu823@126.com. (13) State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China. lsszq@mail.sysu.edu.cn. Institute of Sun Yat-sen University in Shenzhen, Shenzhen, China. lsszq@mail.sysu.edu.cn. (14) Laboratory of Oncology, Center for Molecular Medicine, School of Basic Medicine, Faculty of Medicine, Yangtze University, 1 Nanhuan Road, Jingzhou, 434023, Hubei, China. hongwu_xin@126.com. Department of Biochemistry and Molecular Biology, School of Basic Medicine, Faculty of Medicine, Yangtze University, Jingzhou, 434023, Hubei, China. hongwu_xin@126.com. People's Hospital of Lianjiang, Lianjiang, 524400, Guangdong, China. hongwu_xin@126.com.

Unstimulated apheresis for chimeric antigen receptor manufacturing in pediatric/adolescent acute lymphoblastic leukemia patients

Autologous unstimulated leukapheresis product serves as starting material for a variety of innovative cell therapy products, including chimeric antigen receptor (CAR)-modified T-cells. Although it may be reasonable to assume feasibility and efficiency of apheresis for CAR-T cell manufacture, several idiosyncrasies of these patients warrant their separate analysis: target cells (mononuclear cells [MNC] and T-cells) are relatively few which may instruct the selection of apheresis technology, low body weight, and, hence, low total blood volume (TBV) can restrict process and product volume, and patients may be in compromised health. We here report outcome data from 46 consecutive leukaphereses in 33 unique pediatric patients performed for the purpose of CD19-CAR-T-cell manufacturing. Apheresis targets of 2_10(9) MNC/1_10(9) T-cells were defined by marketing authorization holder specification. Patient weight was 8 to 84_kg; TBV was 0.6 to 5.1 L. Spectra Optia apheresis technology was used. For 23 patients, a single apheresis sufficed to generate enough cells and manufacture CAR-T-cells, the remainder required two aphereses to meet target dose and/or two apheresis series because of production failure. Aphereses were technically feasible and clinically tolerable without serious adverse effects. The median collection efficiencies for MNC and T-cells were 53% and 56%, respectively. In summary, CAR apheresis in pediatric patients, including the very young, is feasible, safe and efficient, but the specified cell dose targets can be challenging in smaller children. Continuous monitoring of apheresis outcomes is advocated in order to maintain quality.

Author Info: (1) Division for Stem Cell Transplantation, Immunology and Intensive Care Medicine, Department for Children and Adolescents, Goethe University, Frankfurt/Main, Germany. (2) Divisio

Author Info: (1) Division for Stem Cell Transplantation, Immunology and Intensive Care Medicine, Department for Children and Adolescents, Goethe University, Frankfurt/Main, Germany. (2) Division for Stem Cell Transplantation, Immunology and Intensive Care Medicine, Department for Children and Adolescents, Goethe University, Frankfurt/Main, Germany. (3) Division for Stem Cell Transplantation, Immunology and Intensive Care Medicine, Department for Children and Adolescents, Goethe University, Frankfurt/Main, Germany. (4) Division for Stem Cell Transplantation, Immunology and Intensive Care Medicine, Department for Children and Adolescents, Goethe University, Frankfurt/Main, Germany. (5) German Red Cross Blood Service Baden-WŸrttemberg-Hessen, Institute Frankfurt/Main, Frankfurt/Main, Germany. (6) German Red Cross Blood Service Baden-WŸrttemberg-Hessen, Institute Frankfurt/Main, Frankfurt/Main, Germany. Institute for Transfusion Medicine and Immunohematology, Goethe University, Frankfurt/Main, Germany. (7) Division for Stem Cell Transplantation, Immunology and Intensive Care Medicine, Department for Children and Adolescents, Goethe University, Frankfurt/Main, Germany. (8) German Red Cross Blood Service Baden-WŸrttemberg-Hessen, Institute Frankfurt/Main, Frankfurt/Main, Germany. Institute for Transfusion Medicine and Immunohematology, Goethe University, Frankfurt/Main, Germany. Division of Hematology, Department of Medicine, University of Washington, Seattle, Washington, USA.

Tumor infiltrating lymphocytes (TIL) therapy in metastatic melanoma: boosting of neoantigen-specific T cell reactivity and long-term follow-up

Treatment of metastatic melanoma with autologous tumor infiltrating lymphocytes (TILs) is currently applied in several centers. Robust and remarkably consistent overall response rates, of around 50% of treated patients, have been observed across hospitals, including a substantial fraction of durable, complete responses. PURPOSE: Execute a phase I/II feasibility study with TIL therapy in metastatic melanoma at the Netherlands Cancer Institute, with the goal to assess feasibility and potential value of a randomized phase III trial. EXPERIMENTAL: Ten patients were treated with TIL therapy. Infusion products and peripheral blood samples were phenotypically characterized and neoantigen reactivity was assessed. Here, we present long-term clinical outcome and translational data on neoantigen reactivity of the T cell products. RESULTS: Five out of 10 patients, who were all anti-PD-1 na•ve at time of treatment, showed an objective clinical response, including two patients with a complete response that are both ongoing for more than 7 years. Immune monitoring demonstrated that neoantigen-specific T cells were detectable in TIL infusion products from three out of three patients analyzed. For six out of the nine neoantigen-specific T cell responses detected in these TIL products, T cell response magnitude increased significantly in the peripheral blood compartment after therapy, and neoantigen-specific T cells were detectable for up to 3 years after TIL infusion. CONCLUSION: The clinical results from this study confirm the robustness of TIL therapy in metastatic melanoma and the potential role of neoantigen-specific T cell reactivity. In addition, the data from this study supported the rationale to initiate an ongoing multicenter phase III TIL trial.

Author Info: (1) BioTherapeutics Unit, Netherlands Cancer Institute, Amsterdam, The Netherlands. (2) Division of Molecular Oncology and Immunology, Netherlands Cancer Institute, Amsterdam, The

Author Info: (1) BioTherapeutics Unit, Netherlands Cancer Institute, Amsterdam, The Netherlands. (2) Division of Molecular Oncology and Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands. (3) Division of Molecular Oncology and Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands. (4) Division of Molecular Oncology and Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands. (5) Division of Molecular Oncology and Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands. (6) BioTherapeutics Unit, Netherlands Cancer Institute, Amsterdam, The Netherlands. (7) BioTherapeutics Unit, Netherlands Cancer Institute, Amsterdam, The Netherlands. (8) BioTherapeutics Unit, Netherlands Cancer Institute, Amsterdam, The Netherlands. (9) Division of Molecular Oncology and Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands. (10) Division of Molecular Oncology and Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands. (11) Division of Molecular Oncology and Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands. (12) AIMM Therapeutics, Amsterdam, The Netherlands. Experimental Immunology, Amsterdam University Medical Centres, Amsterdam, Noord-Holland, The Netherlands. (13) AIMM Therapeutics, Amsterdam, The Netherlands. (14) Department of Medical Oncology, Netherlands Cancer Institute, Amsterdam, The Netherlands. (15) Department of Medical Oncology, Netherlands Cancer Institute, Amsterdam, The Netherlands. (16) Department of Surgery, Leiden Universitair Medisch Centrum, Leiden, Zuid-Holland, The Netherlands. (17) Surgical Oncology, Antoni van Leeuwenhoek Nederlands Kanker Instituut, Amsterdam, The Netherlands. Dutch Institute for Clinical Auditing, Leiden, The Netherlands. (18) Department of Biometrics, Netherlands Cancer Institute, Amsterdam, The Netherlands. (19) Department of Medical Oncology, Netherlands Cancer Institute, Amsterdam, The Netherlands. (20) Department of Medical Oncology, Netherlands Cancer Institute, Amsterdam, Noord-Holland, The Netherlands. (21) Department of Pharmacy & Pharmacology, Netherlands Cancer Institute, Amsterdam, The Netherlands. Division of Pharmacoepidemiology and Clinical Pharmacology, Utrecht University Department of Pharmaceutical Sciences, Utrecht, Utrecht, The Netherlands. (22) Department of Pharmacy & Pharmacology, Netherlands Cancer Institute, Amsterdam, The Netherlands. (23) Division of Molecular Oncology and Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands. Oncode Institute, Utrecht, The Netherlands. (24) Division of Molecular Oncology and Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands j.haanen@nki.nl.

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