Journal Articles

'Omics analyses

Genome, transcriptome, proteome, etc. studies that help to understand and improve cancer immunotherapy

The ins and outs of type I iNKT cell development

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Natural killer T (NKT) cells are innate-like lymphocytes that bridge the gap between the innate and adaptive immune responses. Like innate immune cells, they have a mature, effector phenotype that allows them to rapidly respond to threats, compared to adaptive cells. NKT cells express T cell receptors (TCRs) like conventional T cells, but instead of responding to peptide antigen presented by MHC class I or II, NKT cell TCRs recognize glycolipid antigen in the context of CD1d. NKT cells are subdivided into classes based on their TCR and antigen reactivity. This review will focus on type I iNKT cells that express a semi invariant Valpha14Jalpha18 TCR and respond to the canonical glycolipid antigen, alpha-galactosylceramide. The innate-like effector functions of these cells combined with their T cell identity make their developmental path quite unique. In addition to the extrinsic factors that affect iNKT cell development such as lipid:CD1d complexes, co-stimulation, and cytokines, this review will provide a comprehensive delineation of the cell intrinsic factors that impact iNKT cell development, differentiation, and effector functions - including TCR rearrangement, survival and metabolism signaling, transcription factor expression, and gene regulation.

Author Info: (1) Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St. HSF-1 Room 380, Baltimore, MD 21201, USA. Electronic address

Author Info: (1) Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St. HSF-1 Room 380, Baltimore, MD 21201, USA. Electronic address: susannah.shissler@umaryland.edu. (2) Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St. HSF-1 Room 380, Baltimore, MD 21201, USA.

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Preclinical development of the TLR9 agonist DV281 as an inhaled aerosolized immunotherapeutic for lung cancer: Pharmacological profile in mice, non-human primates, and human primary cells

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CpG-motif-containing oligodeoxynucleotides (CpG-ODN) activate innate immunity through Toll-Like Receptor (TLR) 9 signaling and generate local immune responses when delivered directly to the lung. Herein we describe pharmacological studies in mice, cynomolgus monkeys, and in human primary cells which support the development of DV281, a C-class CpG-ODN, as an inhaled aerosolized immunotherapeutic for lung cancer to be combined with an inhibitor of the anti-programmed cell death protein 1 (PD1) immune checkpoint. In vitro, DV281 potently induced Interferon (IFN)alpha from monkey and human peripheral blood mononuclear cells (PBMCs), stimulated interleukin6 production and proliferation in human B cells, and induced TLR9-dependent cytokine responses from mouse splenocytes. Intranasal delivery of DV281 to mice led to substantial but transient cytokine and chemokine responses in the lung. Lung responses to repeated intranasal DV281 were partially to fully reversible 2weeks after the final dose and were absent in TLR9-deficient mice. Single escalating doses of aerosolized DV281 in monkeys induced dose-dependent induction of IFN-regulated genes in bronchoalveolar lavage cells and blood. In a repeat-dose safety study in monkeys, inhaled DV281 was well-tolerated, and findings were mechanism of action-related and non-adverse. Co-culture of human PBMC with DV281 and anti-PD1 antibody did not augment cytokine or cellular proliferation responses compared to DV281 alone, indicating that the combination did not lead to dysregulated cytokine responses. These studies support clinical development of inhaled aerosolized DV281 as a combination therapy with anti-PD1 antibody for lung cancer immunotherapy.

Author Info: (1) Dynavax Technologies, Berkeley, CA, USA. (2) Dynavax Technologies, Berkeley, CA, USA. (3) Dynavax Technologies, Berkeley, CA, USA. (4) Dynavax Technologies, Berkeley, CA, USA. (5)

Author Info: (1) Dynavax Technologies, Berkeley, CA, USA. (2) Dynavax Technologies, Berkeley, CA, USA. (3) Dynavax Technologies, Berkeley, CA, USA. (4) Dynavax Technologies, Berkeley, CA, USA. (5) Dynavax Technologies, Berkeley, CA, USA. (6) Dynavax Technologies, Berkeley, CA, USA. Electronic address: dcampbell@dynavax.com.

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Reduction of non-specific toxicity of immunotoxin by intein mediated reconstitution on target cells

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Recombinant immunotoxins are chimeric proteins composed of a targeting peptide that binds to a specific tumor antigen and a toxin protein killing target cells. Recombinant immunotoxin exhibits potent cancer inhibiting effects both in vivo and in vitro. However, the non-specific toxicity causes severe syndromes limiting their clinical application. To reduce toxicity caused by recombinant immunotoxins in general, we divided an immunotoxin into two nontoxic segments that may restore toxic bioactivity on tumor cell surface based on the intein mediated trans-splicing reaction. Both split and reconstituted immunotoxins were tested for their biological activities. We found that the reconstituted immunotoxin retained antigen specificity and affinity toward cancer cells overexpressing HER2/neu. After being internalized into HER2/neu positive cells, the reconstituted immunotoxin showed comparable cytotoxicity as the original immunotoxin, while the split immunotoxin fragments showed no toxic activity to cells with or without HER2/neu expression. This approach can potentially be used under clinical settings to reduce non-specific toxicity by administering patients with inactive immunotoxin fragments. Cytotoxic effect only occurs at tumor sites where the inactive fragments bind, trans-splice and become active toxin.

Author Info: (1) Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People's

Author Info: (1) Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People's Republic of China. (2) Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People's Republic of China. (3) Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People's Republic of China. (4) Jecho Laboratories, Inc., 7320 Executive Way, Frederick MD21704, USA. (5) Jecho Laboratories, Inc., 7320 Executive Way, Frederick MD21704, USA. (6) Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People's Republic of China; Jecho Laboratories, Inc., 7320 Executive Way, Frederick MD21704, USA. Electronic address: jianweiz@sjtu.edu.cn.

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Which is the optimal immunotherapy for advanced squamous non-small-cell lung cancer in combination with chemotherapy: anti-PD-1 or anti-PD-L1

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Recent randomized phase III trials (KEYNOTE-407 and IMpower131) reported that adding anti-programmed death (ligand) 1 (anti-PD-(L)1) antibodies in combination with taxane-platinum improve the therapeutic efficacy for advanced squamous non-small-cell lung cancer (NSCLC). However, there is no head-to-head comparison of pembrolizumab (anti-PD-1) plus chemotherapy vs. atezolizumab (anti-PD-L1) plus chemotherapy. Therefore, we performed an indirect comparison to explore the optimal choice of anti-PD-(L)1 treatment for advanced squamous NSCLC in combination with chemotherapy. The clinical outcomes were overall survival (OS), progression-free survival (PFS), objective response rate (ORR) and adverse event (AE). For overall patients, pembrolizumab had significantly superior OS (hazard ratio (HR) with 95% confidence interval, 0.67, 0.47-0.94; P = 0.02) and numerically better PFS (HR, 0.79, 0.60-1.04; P = 0.10) than atezolizumab, while they had similar ORR, all cause AE and grade 3-5 AE. For PD-L1 high patients, pembrolizumab and atezolizumab showed similar OS and PFS. However, for PD-L1 low/negative patients, pembrolizumab had superior OS (HR, 0.43, 0.24-0.76; P < 0.01/ HR, 0.74, 0.40-1.38; P = 0.35) and better PFS (HR, 0.80, 0.51-1.26; P = 0.33/ HR, 0.46, 0.28-0.75; P <0.01) than atezolizumab. Our analysis raises the hypothesis that anti-PD-1 antibody therapy in combination with chemotherapy may have superior efficacy compared to anti-PD-L1 antibody combination for patients with PD-L1 low/negative advanced squamous NSCLC.

Author Info: (1) Department of Medical Oncology, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, Guangdong, China. State Key Laboratory of Oncology in South China

Author Info: (1) Department of Medical Oncology, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, Guangdong, China. State Key Laboratory of Oncology in South China, 651 Dongfeng Road East, Guangzhou, Guangdong, China. Collaborative Innovation Center for Cancer Medicine, 651 Dongfeng Road East, Guangzhou, Guangdong, China. (2) Department of Medical Oncology, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, Guangdong, China. State Key Laboratory of Oncology in South China, 651 Dongfeng Road East, Guangzhou, Guangdong, China. Collaborative Innovation Center for Cancer Medicine, 651 Dongfeng Road East, Guangzhou, Guangdong, China. (3) Department of Medical Oncology, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, Guangdong, China. zhangli6@mail.sysu.edu.cn. State Key Laboratory of Oncology in South China, 651 Dongfeng Road East, Guangzhou, Guangdong, China. zhangli6@mail.sysu.edu.cn. Collaborative Innovation Center for Cancer Medicine, 651 Dongfeng Road East, Guangzhou, Guangdong, China. zhangli6@mail.sysu.edu.cn.

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Immunoregulation effects of TIM-3 on tumors

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Cancer poses a serious threat to human health and the incidence rate has gradually increased, and it has become one of the common causes of death. Immune factors are one of the important factors affecting the survival and expansion of tumor cells in vivo. Cancer patients have obvious cell immune dysfunction and low anti-tumor immunity. TIM-3 can be widely expressed in a variety of immune cells. It can affect the innate immune and adaptive immune response by regulating the function of immune cells, thus affecting the occurrence and development of tumors. This paper focuses on the regulation of TIM-3 on immune cells, and the expression and mechanism in patients with liver cancer, gastric cancer, prostate cancer and so on, in order to explore the regulatory mechanism of TIM-3 in tumor immunity, and to provide new ideas and targets for immunotherapy of tumors.

Author Info: (1) Department of Obstetrics and Gynecology, Shanghai General Hospital , Shanghai Jiaotong University, Shanghai, China. (2) Department of Clinical Laboratory, The Second Hospital of Shandong University, Jinan, China.

Author Info: (1) Department of Obstetrics and Gynecology, Shanghai General Hospital , Shanghai Jiaotong University, Shanghai, China. (2) Department of Clinical Laboratory, The Second Hospital of Shandong University, Jinan, China.

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Midostaurin reduces Regulatory T cells markers in Acute Myeloid Leukemia

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Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy in which the only curative approach is allogeneic stem cell transplant (Allo-HSCT). The recognition and elimination of leukemic clones by donor T-cells contribute significantly to Allo-HSCT success. FLT3-ITD, a common mutation in AML, is associated with poor prognosis. Recently, midostaurin became the first FDA approved FLT3-inhibitor for pre-transplant patients with FLT3-ITD in combination with standard therapy. In addition to their multikinase activity which may affect T-cell signaling, FLT3-inhibitors induce apoptosis of malignant cells which may also enhance antigen presentation to activate T-cells. Considering the increased clinical use of these inhibitors in patients with AML, and the limited clinical benefit derived from their use as single agents, understanding how FLT3-inhibitors affect T cell population and function is needed to improve their clinical benefit. We examined the effect of four different FLT3 inhibitors (midostaurin, sorafenib, tandutinib, and quizartenib) on T cell populations in peripheral blood mononuclear cells (PBMC) obtained from healthy donors and from patients with AML. Midostaurin exhibited a significant decrease in CD4 + CD25 + FOXP3+ T cell population and FOXP3 mRNA expression in healthy and AML PBMCs. Similarly, samples collected from patients with AML treated with midostaurin showed a reduction in Tregs markers. Interferon-gamma(IFN-gamma), tumor necrosis factor-alpha(TNF-alpha), and IL-10 levels were also reduced following midostaurin treatment. Considering the FDA approval of midostaurin for use in patients with AML in the pre-transplant setting, our finding will have important clinical implication as it provides the rationale for functional investigation of the use of midostaurin in post-transplant patients.

Author Info: (1) School of Pharmacy, University of Southern California, Los Angeles, CA, USA. (2) School of Pharmacy, University of Southern California, Los Angeles, CA, USA. (3)

Author Info: (1) School of Pharmacy, University of Southern California, Los Angeles, CA, USA. (2) School of Pharmacy, University of Southern California, Los Angeles, CA, USA. (3) School of Pharmacy, University of Southern California, Los Angeles, CA, USA. (4) Norris Comprehensive Cancer Center, USC, Los Angeles, CA, USA. (5) School of Pharmacy, University of Southern California, Los Angeles, CA, USA. alachkar@usc.edu. Norris Comprehensive Cancer Center, USC, Los Angeles, CA, USA. alachkar@usc.edu.

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Improved Multiplex Immunohistochemistry for Immune Microenvironment Evaluation of Mouse Formalin-Fixed, Paraffin-Embedded Tissues

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Immune profiling of tissue through multiplex immunohistochemistry is important for the investigation of immune cell dynamics, and it can contribute to disease prognosis and evaluation of treatment response in cancer patients. However, protocols for mouse formalin-fixed, paraffin-embedded tissue have been less successful. Given that formalin fixation and paraffin embedding remains the most common preparation method for processing mouse tissue, this has limited the options to study the immune system and the impact of novel therapeutics in preclinical models. In an attempt to address this, we developed an improved immunohistochemistry protocol with a more effective Ag-retrieval buffer. We also validated 22 Abs specific for mouse immune cell markers to distinguish B cells, T cells, NK cells, macrophages, dendritic cells, and neutrophils. In addition, we designed and tested novel strategies to identify immune cells for which unique Abs are currently not available. Last, in the 4T1 model of breast cancer, we demonstrate the utility of our protocol and Ab panels in the quantitation and spatial distribution of immune cells.

Author Info: (1) Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390. (2) Division

Author Info: (1) Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390. (2) Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390. (3) Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390. (4) Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390. (5) Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390. (6) Centre for Cancer Immunology, Faculty of Medicine, Southampton General Hospital, University of Southampton, Southampton 016 6YD, United Kingdom. (7) Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390. (8) Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030; and. (9) Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390; rolf.brekken@utsouthwestern.edu. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390.

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The Balancing Act between Cancer Immunity and Autoimmunity in Response to Immunotherapy

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The explosion in novel cancer immunotherapies has resulted in extraordinary clinical successes in the treatment of multiple cancers. Checkpoint inhibitors (CPIs) that target negative regulatory molecules have become standard of care. However, with the growing use of CPIs, alone or in combination with chemotherapy, targeted therapies, or other immune modulators, a significant increase in immune-related adverse events (irAEs) has emerged. The wide-ranging and currently unpredictable spectrum of CPI-induced irAEs can lead to profound pathology and, in some cases, death. Growing evidence indicates that many irAEs are a consequence of a breakdown in self-tolerance, but the influence of genetics, the environment, and the mechanisms involved remains unclear. This review explores key questions in this emerging field, summarizing preclinical and clinical experiences with this new generation of cancer drugs, the growing understanding of the role of the immune response in mediating these toxicities, the relationship of CPI-induced autoimmunity to conventional autoimmune diseases, and insights into the mechanism of irAE development and treatment.

Author Info: (1) Diabetes Center and Sean N. Parker Autoimmune Research Laboratory, University of California San Francisco, San Francisco, California. QIMR Berghofer Medical Research Institute, Herston, Queensland

Author Info: (1) Diabetes Center and Sean N. Parker Autoimmune Research Laboratory, University of California San Francisco, San Francisco, California. QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia. (2) Diabetes Center and Sean N. Parker Autoimmune Research Laboratory, University of California San Francisco, San Francisco, California. Division of Endocrinology, Department of Medicine, University of California San Francisco, San Francisco, California. (3) Diabetes Center and Sean N. Parker Autoimmune Research Laboratory, University of California San Francisco, San Francisco, California. jbluestone@parkerici.org. Parker Institute for Cancer Immunotherapy, San Francisco, California.

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Tumor-infiltrating lymphocytes: Streamlining a complex manufacturing process

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Adoptive cell therapy of tumor-infiltrating lymphocytes has shown promise for treatment of refractory melanoma and other solid malignancies; however, challenges to manufacturing have limited its widespread use. Traditional manufacturing efforts were lengthy, cumbersome and used open culture systems. We describe changes in testing and manufacturing that decreased the process cycle time, enhanced the robustness of critical quality attribute testing and facilitated a functionally closed system. These changes have enabled export of the manufacturing process to support multi-center clinical trials.

Author Info: (1) Cellular Therapy Core Facility, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA. Electronic address: emlhope@iu.edu. (2) Cellular Therapy Core Facility, H

Author Info: (1) Cellular Therapy Core Facility, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA. Electronic address: emlhope@iu.edu. (2) Cellular Therapy Core Facility, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA. (3) Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA. (4) Cellular Therapy Core Facility, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA; Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.

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A novel tetravalent bispecific antibody targeting programmed death 1 and tyrosine-protein kinase Met for treatment of gastric cancer

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Background Redirecting T cells to tumor cells using bispecific antibodies (BsAbs) is emerging as a potent cancer therapy. The main concept of this strategy is to cross-link tumor cells and T cells by simultaneously binding to cell surface tumor-associated antigen (TAA) and the CD3 chain. However, immune checkpoint programmed cell death ligand-1 (PD-L1) on tumor cells or other myeloid cells upreglulated remarkablely after the treatment of CD3-binding BsAbs, leads to the generation of suppressed microenvironment for immune evasion and tumor progression. Although this resistance could be partially reversed by anti-PD-L1 treatment, targeting two pathways through one antibody-based molecule may provide a strategic advantage over the combination of BsAbs and immune checkpoint inhibitors. Methods We developed two novel BsAbs PD-1/c-Met DVD-Ig and IgG-scFv both targeting PD-1 to restore the immune effector function of T cells and engaging them to tumor cells via binding to cellular-mesenchymal to epithelial transition factor (c-Met). Binding activities, T cell activation and proliferation were analyzed by flow cytometry. Cell Cytotoxicity and cytokine release were measured using LDH release assay and ELISA, respectively. Anti-tumor response in vivo was evaluated by generate xenograft models in NOD-SCID mice. Results These bispecific antibodies exhibited effective antitumor activity against high- and low- c-Met-expressing gastric cancer cell lines in vitro and mediated strong tumor growth inhibition in human gastric cancer xenograft models. Conclusion The engagement of the PD-1/PD-L1 blockade to c-Met-overexpressing cancer cells is a promising strategy for the treatment of gastric cancer and potentially other malignancies.

Author Info: (1) Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fudan University, P.O. Box #238 No. 138 Yi Xue Yuan Road, Shanghai, China. (2)

Author Info: (1) Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fudan University, P.O. Box #238 No. 138 Yi Xue Yuan Road, Shanghai, China. (2) Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fudan University, P.O. Box #238 No. 138 Yi Xue Yuan Road, Shanghai, China. (3) Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fudan University, P.O. Box #238 No. 138 Yi Xue Yuan Road, Shanghai, China. (4) Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fudan University, P.O. Box #238 No. 138 Yi Xue Yuan Road, Shanghai, China. (5) Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fudan University, P.O. Box #238 No. 138 Yi Xue Yuan Road, Shanghai, China. (6) Department of Medical Oncology, Shanghai Cancer Center, School of Basic Medicine, Fudan University, P.O. Box #238 No. 138 Yi Xue Yuan Road, Shanghai, China. wanghj98@hotmail.com. (7) Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fudan University, P.O. Box #238 No. 138 Yi Xue Yuan Road, Shanghai, China. minyu@shmu.edu.cn.

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