Adoptive Cell Transfer Immunotherapy Research Articles

FLT3 signaling augments macrophage production from human pluripotent stem cells.

Recent advances in cell engineering technologies enable immune cells to be utilized for adoptive cell transfer (ACT) immunotherapy against cancers. Macrophages have the potential to directly and indirectly exterminate cancers and are therefore an attractive option for therapies. To develop new ACT therapies using macrophages, a great number of macrophages are required. Human induced pluripotent stem cells (iPSCs) are expected to be a source of macrophages; therefore, a system to efficiently produce macrophages from human iPSCs is needed. Here, we demonstrated that human iPSCs were robustly differentiated into macrophages by enforced FMS-like tyrosine kinase-3 (FLT3) signaling via the introduction of exogenous FLT3 into iPSCs and the addition of its ligand FLT3L to the macrophage induction culture. These iPSC-derived macrophages were identical to those obtained by standard differentiation induction methods. Thus, our novel system enables the preparation of scalable macrophages from human iPSCs. We believe that this system will be useful to develop a novel ACT therapy using macrophages.

Innovative Strategies of Reprogramming Immune System Cells by Targeting CRISPR/Cas9-Based Genome-Editing Tools: A New Era of Cancer Management.

The recent developments in the study of clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) system have revolutionized the art of genome-editing and its applications for cellular differentiation and immune response behavior. This technology has further helped in understanding the mysteries of cancer progression and possible designing of novel antitumor immunotherapies. CRISPR/Cas9-based genome-editing is now often used to engineer universal T-cells, equipped with recombinant T-cell receptor (TCR) or chimeric antigen receptor (CAR). In addition, this technology is used in cytokine stimulation, antibody designing, natural killer (NK) cell transfer, and to overcome immune checkpoints. The innovative potential of CRISPR/Cas9 in preparing the building blocks of adoptive cell transfer (ACT) immunotherapy has opened a new window of antitumor immunotherapy and some of them have gained FDA approval. The manipulation of immunogenetic regulators has opened a new interface for designing, implementation and interpretation of CRISPR/Cas9-based screening in immuno-oncology. Several cancers like lymphoma, melanoma, lung, and liver malignancies have been treated with this strategy, once thought to be impossible. The safe and efficient delivery of CRISPR/Cas9 system within the immune cells for the genome-editing strategy is a challenging task which needs to be sorted out for efficient immunotherapy. Several targeting approaches like virus-mediated, electroporation, microinjection and nanoformulation-based methods have been used, but each procedure offers some limitations. Here, we elaborate the recent updates of cancer management through immunotherapy in partnership with CRISPR/Cas9 technology. Further, some innovative methods of targeting this genome-editing system within the immune system cells for reprogramming them, as a novel strategy of anticancer immunotherapy is elaborated. In addition, future prospects and clinical trials are also discussed.

T-lymphocytes from focused ultrasound ablation subsequently mediate cellular antitumor immunity after adoptive cell transfer immunotherapy.

Our previous studies found that high-intensity focused ultrasound (HIFU) stimulated tumor-specific T cells in a mouse H22 tumor model, and adoptive transfer of the T cells from HIFU-treated mice could subsequently elicit stronger inhibition on the growth and progression of the implanted tumors. The aim of this study was to investigate the mechanism of T cells from focused ultrasound ablation in HIFU-mediated immunomodulation. Sixty H22 tumor-bearing mice were treated by either HIFU or sham-HIFU, and 30 naïve syngeneic mice served as controls. All mice were euthanized on day 14 after HIFU and splenic T cell suspensions were obtained in each group. Using an adoptive cell transfer model, a total of 1 × 106 T cells from HIFU treated-mice were intravenously injected into each syngeneic H22 tumor-bearing mouse twice on day 3 and 4, followed by the sacrifice for immunological assessments at 14 days after the adoptive transfer. T cells from HIFU-treated mice could significantly enhance the cytotoxicity of CTLs (p < 0.001), with a significant increase of TNF-α (p < 0.001) and IFN-γ secretion (p < 0.001). Compared to control and sham-HIFU groups, the number of Fas ligand+ and perforin+ tumor-infiltrating lymphocytes (TILs) and apoptotic H22 tumor cells were significantly higher (p < 0.001) in the HIFU group. There were linear correlations between apoptotic tumor cells and Fas ligand+ TILs (r = 0.9145, p < 0.001) and perforin+ TILs (r = 0.9619, p < 0.001). T cells from HIFU-treated mice can subsequently mediate cellular antitumor immunity, which may play an important role in the HIFU-based immunomodulation.

Adoptive cell transfer immunotherapy for patients with solid epithelial cancers

Efforts to apply adoptive cell transfer (ACT) immunotherapy to patients with common epithelial cancers have been stimulated by the demonstration that the majority of these patients contain lymphocytes reactive against the expressed products of their cancer mutations. Early efforts to specifically target these antigens have been promising.

Trends in Melanoma Phase 3 Clinical Trials since 2010: Is there Hope for Advanced Melanoma Therapies beyond Approved Treatment Mechanisms?

Several drugs and treatment modalities are under investigation to improve current melanoma therapy options. This review profiles the trends in clinical trial investment in late-stage melanoma, and anticipates what changes are expected in melanoma treatment, with a focus on exploratory drug mechanisms. We reviewed nine international clinical trial databases for registered, interventional, and phase 3 cutaneous melanoma clinical trials since 2010. 73 trials studied drug therapies in late-stage (stage III and IV) melanoma. Exploratory mechanisms were investigated in 32% (23/73) of the late-stage melanoma drug therapy trials. Most exploratory drug trials include immunotherapy drug mechanisms (15/23 trials). Two exploratory mechanisms showed promise: the anti-LAG3 antibody, relatlimab, and the hapten modified vaccine, MVax. Many (52%) trials of exploratory mechanisms are ongoing including the use of adoptive cell transfer immunotherapies, dendritic cell vaccine therapy, and histone deacetylase (HDAC) inhibitors, among others. Since most clinical trials focus on previously approved drug mechanisms, it is likely that paradigm-changing treatments will involve these therapies being used in new treatment contexts or combinations. Only 2 exploratory drug mechanisms studied since 2010 have achieved promising results in the phase 3 setting, though many other trials are ongoing at this time.

Na+/H+-exchanger 1 enhances antitumor activity of engineered NK-92 natural killer cells.

Adoptive cell transfer (ACT) immunotherapy has remarkable efficacy against some hematological malignancies. However, its efficacy in solid tumors is limited by the adverse tumor microenvironment (TME) conditions, most notably that acidity inhibits T and natural killer (NK) cell mTOR complex 1 (mTORC1) activity and impairs cytotoxicity. In several reported studies, systemic buffering of tumor acidity enhanced the efficacy of immune checkpoint inhibitors. Paradoxically, we found in a c-Myc-driven hepatocellular carcinoma model that systemic buffering increased tumor mTORC1 activity, negating inhibition of tumor growth by anti-PD1 treatment. Therefore, in this proof-of-concept study, we tested the metabolic engineering of immune effector cells to mitigate the inhibitory effect of tumor acidity while avoiding side effects associated with systemic buffering. We first overexpressed an activated RHEB in the human NK cell line NK-92, thereby rescuing acid-blunted mTORC1 activity and enhancing cytolytic activity. Then, to directly mitigate the effect of acidity, we ectopically expressed acid extruder proteins. Whereas ectopic expression of carbonic anhydrase IX (CA9) moderately increased mTORC1 activity, it did not enhance effector function. In contrast, overexpressing a constitutively active Na+/H+-exchanger 1 (NHE1; SLC9A1) in NK-92 did not elevate mTORC1 but enhanced degranulation, target engagement, in vitro cytotoxicity, and in vivo antitumor activity. Our findings suggest the feasibility of overcoming the inhibitory effect of the TME by metabolically engineering immune effector cells, which can enhance ACT for better efficacy against solid tumors.

Current Immunotherapeutic Approaches for Malignant Gliomas.

Glioblastoma is the most common malignant central nervous system (CNS) tumor (48.3%), with a median survival of only about 14.6 months. Although the CNS is an immune-privileged site, activated T cells can cross the blood-brain barrier. The recent successes of several immunotherapies for various cancers have drawn interest in immunotherapy for treatment of malignant glioma. There have been extensive attempts to evaluate the efficiency of immunotherapy against malignant glioma. Passive immunotherapy for malignant glioma includes monoclonal antibody-mediated immunotherapy, cytokine-mediated therapy, and adoptive cell transfer, also known as chimeric antigen receptor T cell treatment. On the other hand, active immunotherapy, which stimulates the patient’s adaptive immune system against specific tumor-associated antigens, includes cancer vaccines that are divided into peptide vaccines and cell-based vaccines. In addition, there is immune checkpoint blockade therapy, which increases the efficiency of immunotherapy by reducing the resistance of malignant glioma to immunotherapy. Despite centuries of efforts, immunotherapeutic successes for malignant glioma remain limited. However, many clinical trials of adoptive cell transfer immunotherapy on malignant glioma are ongoing, and the outcomes are eagerly awaited. In addition, although there are still several obstacles, current clinical trials using personalized neoantigen-based dendritic cell vaccines offer new hope to glioblastoma patients. Furthermore, immune checkpoint targeted therapy is expected to decipher the mechanism of immunotherapy resistance in malignant glioma in the near future. More studies are needed to increase the efficacy of immunotherapy in malignant glioma. We hope that immunotherapy will become a new treatment of malignant glioma.

Chimeric antigen receptor T cell: A cancer immunotherapy.

During present decade, targeted drug therapy has been the epitome for treatment of cancer. Drugs like Imatinib, a tyrosine kinase receptor inhibitor and Trastuzumab, an human epidermal growth factor receptor-2/neu inhibitor, has been developed and accepted widely for management of chronic myeloid leukaemia and breast cancer respectively. Recent development among the various immunotherapies is adoptive cell transfer (ACT). Research on development of various types of ACT immunotherapy is going on, but so far, Chimeric antigen receptors T cell therapy (CAR-T) has achieved the maximum advancement in terms of clinical development. CARs are the modified receptors that integrates specificity and responsiveness onto immune cells to enhance the recognition of cancer cells. For the CAR-T, the T cells are sequestered from a blood of a participant via apheresis. DNA of particular antigen is injected into harvested T cells to generate CARs on cell surface. Following surface manifestation of receptors, multiplication is carried out in enriched media followed by infusion into patient. After infusion, CAR-T cells targeted and exterminate the cancer cells. Initially, only two drugs targeting CD19 as genetically modified autologous immunotherapy has been approved in CAR-T therapy i.e., Tisagenlecleucel and Axicabtagene Ciloleucel, which are discussed in detail in current review. Recently two more drugs got approval i.e., brexucabtagene ciloleucel and lisocabtagene maraleucel, both are directed against CD19, similar to tisagenlecleucel. CAR-T cell therapy is approved for management of Acute Lymphoblastic Leukaemia, Chronic Lymphocytic Leukaemia and lymphoma. CAR-T cell persistence responsible for effectiveness and safety concerns are barriers for their wide application among patients. Growth factor receptors and cluster of differentiation are new drugs targets that are being explored as effective immunotherapy against cancers.

47MO Metabolic intervention during CAR T-cell manufacturing improves persistence and antitumor efficacy

Adoptive cell transfer (ACT) immunotherapy is showing impressive responses in hematological cancers and in metastatic melanoma. However, both the magnitude of tumor responses and the fraction of patients benefitting from this novel therapeutic approach remains limited. T lymphocytes prepared for ACT are generally terminally differentiated, resulting in inefficient engraftment. It has been shown that the infusion of T cells with a self-renewing, memory phenotype confers a stronger and more sustained anti-tumor response.

Renal Cell Carcinoma-Infiltrating CD3low Vγ9Vδ1 T Cells Represent Potentially Novel Anti-Tumor Immune Players.

Due to the highly immunogenic nature of renal cell carcinoma (RCC), the tumor microenvironment (TME) is enriched with various innate and adaptive immune subsets. In particular, gamma-delta (γδ) T cells can act as potent attractive mediators of adoptive cell transfer immunotherapy because of their unique properties such as non-reliance on major histocompatibility complex expression, their ability to infiltrate human tumors and recognize tumor antigens, relative insensitivity to immune checkpoint molecules, and broad tumor cytotoxicity. Therefore, it is now critical to better characterize human γδ T-cell subsets and their mechanisms in RCCs, especially the stage of differentiation. In this study, we aimed to identify γδ T cells that might have adaptive responses against RCC progression. We characterized γδ T cells in peripheral blood and tumor-infiltrating lymphocytes (TILs) in freshly resected tumor specimens from 20 RCC patients. Furthermore, we performed a gene set enrichment analysis on RNA-sequencing data from The Cancer Genome Atlas (TCGA) derived from normal kidneys and RCC tumors to ascertain the association between γδ T-cell infiltration and anti-cancer immune activity. Notably, RCC-infiltrating CD3low Vγ9Vδ1 T cells with a terminally differentiated effector memory phenotype with up-regulated activation/exhaustion molecules were newly detected as predominant TILs, and the cytotoxic activity of these cells against RCC was confirmed in vitro. In an additional analysis of the TCGA RCC dataset, γδ T-cell enrichment scores correlated strongly with those for CTLs, Th1 cells, “exhausted” T cells, and M1 macrophages, suggesting active involvement of γδ T cells in anti-tumor rather than pro-tumor activity, and Vδ1 cells were more abundant than Vδ2 or Vδ3 cells in RCC tumor samples. Thus, we posit that Vγ9Vδ1 T cells may represent an excellent candidate for adoptive immunotherapy in RCC patients with a high risk of relapse after surgery.

Recent advances in immunotherapy, immunoadjuvant, and nanomaterial-based combination immunotherapy

• Different types of cancer immunotherapies and their limitations are reviewed. • Major immunoadjuvants and their limitations treatment are summarized. • The role of nanomaterials in overcoming drawbacks of immunotherapies is highlighted. • Recent advances in nanomaterial-based combination cancer immunotherapy are reviewed. • The outlook of nanomaterial-based combination immunotherapy is presented. Cancer immunotherapy has emerged as the fourth major treatment modality for cancer in addition to chemotherapy, radiotherapy, and surgery. There are various types of cancer immunotherapies that can be generally divided into five categories, namely vaccine immunotherapy, cytokine immunotherapy, checkpoint blockade immunotherapy, adoptive cell transfer immunotherapy, and small molecule immunotherapy. In this review, we describe these therapeutic approaches along with their limitations. Since many immunotherapies rely on immunoadjuvants and nanoplatforms, we summarize different classes of immunoadjuvants used in cancer immunotherapies, including toll-like receptor agonists, plant-derived immunoadjuvants, aluminum salt adjuvants, and other immunoadjuvants. Finally, we provide an in-depth description of nanomaterial-based approaches for the combination of immunotherapy with chemotherapy, phototherapy, radiotherapy, as well as other therapies.

Ocular Outcomes after Treatment of Cytomegalovirus Retinitis Using Adoptive Immunotherapy with Cytomegalovirus-Specific Cytotoxic T Lymphocytes

To describe ocular outcomes in eyes with cytomegalovirus (CMV) retinitis treated with adoptive immunotherapy using systemic administration of CMV-specific cytotoxic Tlymphocytes (CMV-specific CTLs). Retrospective cohort study. Patients with active CMV retinitis evaluated at a tertiary care academic center. Treatment of CMV retinitis with standard-of-care therapy (systemic or intravitreal antivirals) or CMV-specific CTLs (with or without concurrent standard-of-care therapies). The electronic medical record was reviewed to determine baseline characteristics, treatment course, and ocular outcomes, including best-corrected visual acuity (BCVA), treatments administered (CMV-specific CTLs, systemic antivirals, intravitreal antivirals), resolution of CMV retinitis, any occurrence of immune recovery uveitis, cystoid macular edema, retinal detachment, or a combination thereof. Seven patients (3 of whom had bilateral disease [n= 10 eyes]) were treated with CMV-specific CTLs, whereas 20 patients (6 of whom had bilateral disease [n= 26 eyes]) received standard-of-care treatment. Indications for CMV-specific CTL therapy included persistent or progressive CMV retinitis (71.4% of patients); CMV UL54 or UL97 antiviral resistance mutations (42.9%); side effects or toxicity from antiviral agents (57.1%); patient intolerance to longstanding, frequent antiviral therapy for persistent retinitis (28.6%); or a combination thereof. Two patients (28.6%; 4 eyes [40%]) received CMV-specific CTL therapy without concurrent systemic or intravitreal antiviral therapy for active CMV retinitis, whereas 5 patients (71.4%; 6 eyes [60%]) continued to receive concurrent antiviral therapies. Resolution of CMV retinitis was achieved in 9 eyes (90%) treated with CMV-specific CTLs, with BCVA stabilizing (4 eyes [40%]) or improving (4 eyes [40%]) in 80% of eyes over an average follow-up of 33.4 months. Rates of immune recovery uveitis, new-onset cystoid macular edema, and retinal detachment were 0%, 10% (1 eye), and 20% (2 eyes), respectively. These outcomes compared favorably with a nonrandomized cohort of eyes treated with standard-of-care therapy alone, despite potentially worse baseline characteristics. CMV-specific CTL therapy may represent a novel monotherapy or adjunctive therapy, or both, for CMV retinitis, especially in eyes that are resistant, refractory, or intolerant of standard-of-care antiviral therapies. More generally, adoptive cell transfer and adoptive immunotherapy may have a role in refractory CMV retinitis. Larger prospective, randomized trials are necessary.

Label-free inertial-ferrohydrodynamic cell separation with high throughput and resolution.

Rapid and label-free separation of target cells from biological samples provided unique opportunity for disease diagnostics and treatment. However, even with advanced technologies for cell separation, the limited throughput, high cost and low separation resolution still prevented their utility in separating cells with well-defined physical features from a large volume of biological samples. Here we described an ultrahigh-throughput microfluidic technology, termed as inertial-ferrohydrodynamic cell separation (inertial-FCS), that rapidly sorted through over 60 milliliters of samples at a throughput of 100 000 cells per second in a label-free manner, differentiating the cells based on their physical diameter difference with ∼1-2 μm separation resolution. Through the integration of inertial focusing and ferrohydrodynamic separation, we demonstrated that the resulting inertial-FCS devices could separate viable and expandable circulating tumor cells from cancer patients' blood with a high recovery rate and high purity. We also showed that the devices could enrich lymphocytes directly from white blood cells based on their physical morphology without any labeling steps. This label-free method could address the needs of high throughput and high resolution cell separation in circulating tumor cell research and adoptive cell transfer immunotherapy.

Oncolytic Adenovirus in Cancer Immunotherapy.

Simple SummaryOncolytic adenoviruses are engineered to selectively replicate in and destroy cancer tissue. Moreover, these viruses are promising tools to restore antitumor immune response in cancer patients due to their high immunogenicity and the ability to interfere with the immunosuppressive tumor microenvironment. Due to these characteristics, oncolytic adenoviruses can activate tumors for already existing, systemic immunotherapies. The goal of this review is to provide an introduction into the common concepts of oncolytic adenoviruses, and to present their current status in clinical development. We also want to report in detail on strategies to optimize the immunoactivating properties of these agents for future application in multistage cancer immunotherapies.Tumor-selective replicating “oncolytic” viruses are novel and promising tools for immunotherapy of cancer. However, despite their first success in clinical trials, previous experience suggests that currently used oncolytic virus monotherapies will not be effective enough to achieve complete tumor responses and long-term cure in a broad spectrum of cancers. Nevertheless, there are reasonable arguments that suggest advanced oncolytic viruses will play an essential role as enablers of multi-stage immunotherapies including established systemic immunotherapies. Oncolytic adenoviruses (oAds) display several features to meet this therapeutic need. oAds potently lyse infected tumor cells and induce a strong immunogenic cell death associated with tumor inflammation and induction of antitumor immune responses. Furthermore, established and versatile platforms of oAds exist, which are well suited for the incorporation of heterologous genes to optimally exploit and amplify the immunostimulatory effect of viral oncolysis. A considerable spectrum of functional genes has already been integrated in oAds to optimize particular aspects of immune stimulation including antigen presentation, T cell priming, engagement of additional effector functions, and interference with immunosuppression. These advanced concepts have the potential to play a promising future role as enablers of multi-stage immunotherapies involving adoptive cell transfer and systemic immunotherapies.

Strategies to Enhance the Efficacy of T-Cell Therapy for Central Nervous System Tumors.

Mortality rates in patients diagnosed with central nervous system (CNS) tumors, originating in the brain or spinal cord, continue to remain high despite the advances in multimodal treatment regimens, including surgery, radiation, and chemotherapy. Recent success of adoptive cell transfer immunotherapy treatments using chimeric antigen receptor (CAR) engineered T cells against in chemotherapy resistant CD19 expressing B-cell lymphomas, has provided the foundation for investigating efficacy of CAR T immunotherapies in the context of brain tumor. Although significant efforts have been made in developing and translating the novel CAR T therapies for CNS tumors, including glioblastoma (GBM), researchers are yet to achieve a similar level of success as with liquid malignancies. In this review, we discuss strategies and considerations essential for developing robust preclinical models for the translation of T cell-based therapies for CNS tumors. Some of the key considerations include route of delivery, increasing persistence of T cells in tumor environment, remodeling of myeloid environment, establishing the window of treatment opportunity, harnessing endogenous immune system, designing multiple antigen targeting T cells, and rational combination of immunotherapy with the current standard of care. Although this review focuses primarily on CAR T therapies for GBM, similar strategies, and considerations are applicable to all CNS tumors in general.

18F-FHBG PET-CT Reporter Gene Imaging of Adoptive CIK Cell Transfer Immunotherapy for Breast Cancer in a Mouse Model.

BackgroundTo further improve the efficiency of adoptively transferred cytokine-induced killer (CIK) cell immunotherapy in breast cancer (BC), a reliable imaging method is required to visualize and monitor these transferred cells in vivo.MethodsHerpes simplex virus 1-thymidine kinase (HSV1-TK) and 9-(4-[18F]fluoro-3-(hydroxymethyl)butyl)guanine (18F-FHBG) were used as a pair of reporter gene/reporter probe for positron emission tomography (PET) imaging in this study. Following the establishment of subcutaneous BC xenograft-bearing nude mice models, induced human CIK cells expressing reporter gene HSV1-TK through lentiviral transduction were intravenously injected to nude mice. γ-radioimmunoassay was used to determine the specific uptake of 18F-FHBG by these genetically engineered CIK cells expressing HSV1-TK in vitro, and 18F-FHBG micro positron emission tomography-computed tomography (PET-CT) imaging was performed to visualize these adoptively transferred CIK cells in tumor-bearing nude mice.ResultsSpecific uptake of 18F-FHBG by CIK cells expressing HSV1-TK was clearly observed in vitro. Consistently, the localization of adoptively transferred CIK cells in tumor target could be effectively visualized by 18F-FHBG micro PET-CT reporter gene imaging.ConclusionPET-CT reporter gene imaging using 18F-FHBG as a reporter probe enables the visualization and monitoring of adoptively transferred CIK cells in vivo.

Antigen processing and presentation in cancer immunotherapy.

BackgroundKnowledge about and identification of T cell tumor antigens may inform the development of T cell receptor-engineered adoptive cell transfer or personalized cancer vaccine immunotherapy. Here, we review antigen processing and presentation and discuss limitations in tumor antigen prediction approaches.MethodsOriginal articles covering antigen processing and presentation, epitope discovery, and in silico T cell epitope prediction were reviewed.ResultsNatural processing and presentation of antigens is a complex process that involves proteasomal proteolysis of parental proteins, transportation of digested peptides into the endoplasmic reticulum, loading of peptides onto major histocompatibility complex (MHC) class I molecules, and shuttling of peptide:MHC complexes to the cell surface. A number of T cell tumor antigens have been experimentally validated in patients with cancer. Assessment of predicted MHC class I binding and total score for these validated T cell antigens demonstrated a wide range of values, with nearly one-third of validated antigens carrying an IC50 of greater than 500 nM.ConclusionsAntigen processing and presentation is a complex, multistep process. In silico epitope prediction techniques can be a useful tool, but comprehensive experimental testing and validation on a patient-by-patient basis may be required to reliably identify T cell tumor antigens.

Abstract 225: Tumor-infiltrating CD8-positive T-lymphocytes in tubo-ovarian high-grade serous cancer are associated with multiple germline variants in 22q12.1 in a genome-wide association analysis

Abstract Tubo-ovarian high-grade serous cancer (HGSC) is the most common histotype of epithelial ovarian cancer (70%-80%) and the leading cause of death from gynecological malignancy. Novel monoclonal antibody and adoptive cell transfer immunotherapies have found success in cancer in the past five years; however success in HGSC treatment has been elusive thus far. We have recently shown that CD8+ T-lymphocytes infiltrating the tumor proper (TILs) associate in a dose response manner with longer patient survival time regardless of age, stage or residual disease. As understanding host factors associated with this clinically relevant tumor phenotype will contribute to the development of targeted therapies, we sought to identify germline genetic factors associated with CD8+ TIL levels in HGSC. We performed a germline genome-wide association study (GWAS) of CD8+ TILs using 1204 HGSC patients of European ancestry from twelve studies with centralized genotyping, immunohistochemistry, and harmonization of clinical data. Germline genotypes were assayed using the Illumina Infinium OncoArray Beadchip and were imputed to over 11.4 million variants based on the 1000 Genomes Project reference panel. CD8 immunohistochemistry was performed on tissue microarrays using the Leica Bond Rx stainer and scored into four levels of CD8+ TILs per x400 magnification of a 0.55-mm diameter field for each tumor hotspot: none (no CD8+ TILs), low (1-2 CD8+ TILs), moderate (3-19 CD8+ TILs), high (20 or more CD8+ TILs). GWAS used multinomial regression modelling of these four CD8+ TIL levels adjusted for study site, age, and European ancestry principal components. On the average, CD8+ TIL levels were 18%, 19%, 44% and 18% for none, low, moderate and high levels, respectively. As expected, CD8+ TIL levels associated with overall survival time (p &amp;lt; 2.6x10-8), and CD8+ TIL levels and age at diagnosis were similarly distributed across study sites. We identified a region on chromosome 22 with multiple, correlated variants (MAF &amp;gt; 20%) associated with CD8+ TIL levels at p-value &amp;lt; 2.0x10-5. The most statistically significant variant was an indel (rs557925408) located in the an intron of the MYO18B gene (Myosin XVIIIB, p-value &amp;lt; 8.7x10-6). Each copy of this variant associated with a reduced extent of CD8+ TIL infiltration (OR low v none = 0.92; OR moderate v none = 0.61). An independent set of 1200 OncoArray genotyped HGSC cases are undergoing CD8+ TIL scoring in December 2017 and will be included in final genome-wide analysis. This will provide improved statistical power and enable consideration of additional genetic models (e.g., linear trend tests). Comprehensive integrative analysis of germline, tumor, and clinical features provides a model for clinical molecular epidemiology studies and will be key to increasing our understanding of this important HGSC immunophenotype. Citation Format: Yanina Natanzon, Martin Köbel, Stacey J. Winham, Sebastian M. Armasu, Bryan M. McCauley, Robert A. Vierkant, Julie M. Cunningham, David Bowtell, Ian G. Campbell, Jenny Chang-Claude, Anna deFazio, Peter A. Fasching, Mark T. Goodman, Beth Y. Karlan, Francesmary Modugno, Kirsten B. Moysich, Roberta B. Ness, Weiva Sieh, Paul D. Pharoah, Susan J. Ramus, Ellen L. Goode. Tumor-infiltrating CD8-positive T-lymphocytes in tubo-ovarian high-grade serous cancer are associated with multiple germline variants in 22q12.1 in a genome-wide association analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 225.

Reenergized Adoptive Cell Transfer Immunotherapy

Immunotherapy, in treating solid tumors, has seen increased efficacy due to the development of novel strategies of implementation. One innovative strategy, in development by Dr. Weiguo Cui and his colleagues, combines adoptive cell transfer (ACT) with pathogen‐based vaccine techniques in what is called Reenergized ACT (ReACT). Tumor specific CD8 T‐Cells that already have a tumor specific T‐Cell Receptor (TCR), are genetically modified outside the body to express a second TCR that will recognize a bacterial antigen. These newly created, dual specific T cells, when introduced to mice in combination with intratumoral injection of bacterial vaccine, show a significant decrease in tumor volume than their mono‐specific T‐Cell counterparts. The Marquette High SMART (Students Modeling a Research Topic) Team used 3D printing technology, in partnership with the Milwaukee School of Engineering Center for Biomolecular Modeling, to model the TCR complex in order to study its structure and function to better understand ReACT. TCR is a heterodimeric protein consisting of highly variable alpha and beta chains. Both chains are composed of a variable region and a constant region, both of which form antiparallel beta sheets. The variable region binds to the MHC complex whereas the constant region is near the cell membrane of the T‐cell. The constant domains of the TCR consists of cysteine residues that form disulfide bonds, which form a link between the two chains. Further research into immunotherapy, TCR structure and function, and the implementation of Reenergized ACT has major promise in increasing the efficacy of treating disease in human beings now and in the future to come.Support or Funding InformationWeiguo Cui, MD, PhD at the Blood Research Institute, BloodCenter of Wisconsin &amp; Medical College of WisconsinDiane Muzenmaier at the Milwaukee School of Engineering Center for Biomolecular ModelingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A kinetic investigation of interacting, stimulated T cells identifies conditions for rapid functional enhancement, minimal phenotype differentiation, and improved adoptive cell transfer tumor eradication.

For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion in vivo through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell—T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8+ and CD4+ T cells collected from a patient with metastatic melanoma.