Cells in their natural environment are surrounded by a complex extracellular matrix that provides structure and functional cues to the surrounding cells. Hydrogels can be used to approximate this environment for 3D cell culture, but require preparative work including hydrogel assembly, hydrogel optimization, and cell encapsulation. Learn how our fully ready-to-use TrueGel3D™ HTS hydrogel plates help you to quickly and easily establish 3D cell cultures for high-throughput screening and high content analysis. Data will be presented to show how these automation-compatible plates can be used for imaging 3D cultures in immuno-oncology, angiogenesis, neuroscience, and ADME/tox studies.

Learning Objectives:

1. Understand how TrueGel3D® HTS plates solve current limitations of 3D cell cultures for drug discovery.

2. Know how to easily establish cocultures of tumor spheroids and T-cells in 3D using TrueGel3D® HTS plates

3. Gain insight into performing high-throughput drug screen using TrueGel3D® HTS plates

Unravelling the expression, distribution and interaction of tumoral markers is crucial for disease diagnostic and treatment. The past decade marks a milestone in the field of tissue analytics. From low- to high-multiplexing methods of immunohistochemistry (IHC), new technologies have led to the identification of complex patterns of tumoral markers Our presentation will provide an overview of how we could upgrade your standard IHC methods to multiplexing IHC methods as well as spatial genomics technic for single-transcript detection. We will also introduce how automation can support the quality, efficiency, and rapidity of all these technics.

Learning Objectives:

1. Discover how a novel high-precision microfluidic technology enables rapid automated tissue analysis

2. Learn how a microfluidic-based instrument allow for protein and acid nucleic study

The WOLF Cell Sorter from NanoCellect Biomedical was designed to provide an affordable microfluidic benchtop solution to sort cell easily, gently using sterile microfluidic cartridges. NanoCellect is launching the WOLF G2 cell sorter, a 2-laser system with up to 9 fluorescent channels to enable a broader range of applications. During this presentation, we will review several case studies highlighting how the WOLF can use intuitive multi-color sorting strategy to isolate sensitive cells such as neutrophils and improve sample preparation of PBMCs for 10x Genomics single-cell RNA-Seq. 

Learning Objectives:

PD-L1 is a key inhibitor of T cell activation that is often over-expressed in cancer to escape immune surveillance and promote tumor progression.  Blocking antibodies against PD-L1 or its receptor, PD-1, have shown significant clinical benefit in some patients with PD-L1 expressing tumors. Hence, there is great interest in generating therapeutic antibodies against these targets to counteract the immune suppression mechanism that tumors rely on for survival. Most of the anti-PD-L1 therapies in the clinic have been generated by standard hybridoma technology. We investigated whether superior anti-PD-L1 antibodies with greater diversity, affinity, and/or functional activity could be generated using single B cell cloning (BCC) which could circumvent a labor-intensive and time consuming process. Thus, we generated unique antibodies against PD-L1 via both methods. The Beacon® platform (Berkeley Lights, Inc., (BLI)) enabled screening of 33,377 antibody secreting plasma B cells (PCs) against PD-L1.  After penning CD138+ single PCs onto OptoSelectTM chips, we identified over 200 antibodies with binding to PD-L1, and of those, 35 antibodies blocked binding of PD-1 to PD-L1.  The hits were exported for antibody sequence recovery from single B cells and 24 hu-IgG4 chimeric antibodies were generated. For the traditional hybridoma approach, Balb/c and SJL mice were immunized with recombinant PD-L1 and 13,536 hybridoma cells were screened. We purified 44 hybridoma antibodies and 24 BCC antibodies which were characterized for FACS binding, receptor blocking, epitope binning & affinity measurements. Image based selection in the blocking assay enabled identification of 58% true blockers by Beacon with IC50s comparable to benchmarks. We found that 2 antibodies from Beacon and 3 antibodies from hybridoma had triple-digit pM affinity. Taken together, our data show that plasma B cells secreting functional antibody candidates can be rapidly identified on Beacon compared to several months for a hybridoma campaign, thus substantially accelerating the antibody discovery process.  Although the hybridoma approach allowed recovery of a greater number of blocking antibodies, B cell cloning on the Beacon platform enables greater access to the immune repertoire coupled with the ability to screen through robust functional assays up front, thus enabling identification of high affinity, potent anti-PD-L1 antibodies.  

Learning Objectives:

1. Functional assessment of clones on Beacon platform versus Hybridoma technology

2. Characterization of clones discovered by both methods

3. Comparison of clones by both methods against Benchmark antibodies

Hypoxia is pervasive across human cancer and its association with poor outcomes and aggressive malignant phenotypes has been increasingly appreciated.  Previously, we and others described how T cells are virtually absent from hypoxic zones of tumors. Serendipitously, we discovered that the hypoxia-activated prodrug (HAP) TH-302 could potently reduce hypoxia in murine models of prostate, pancreatic and head and neck cancer. With this tool, we were able to test the hypothesis that reversal of hypoxia might restore T cell infiltration and improve sensitivity to T cell checkpoint blockade in “cold” tumors.  Across both transplantable and spontaneous murine models of prostate cancer, we have shown that the combination of TH-302 and antibody blockade of CTLA-4 and PD-1 synergize in curing, regressing, or controlling these tumors.  With our collaborator Dr. Hong, we launched a clinical trial of the combination of TH-302 (evofosfamide) and ipilimumab (αCTLA-4) in 2017 (NCT03098160) which has revealed the potential of this combination to regress established metastatic prostate and head and neck cancers in patients, and to significantly extend progression-free survival in late stage pancreatic cancer.  While TH-302 can restore tumor oxygen supply, we have observed across multiple human and murine cancers that elevated oxygen metabolism (OxPhos) at the tumor cell level metabolically cripples T cells resulting in resistance to immunotherapy.  Ongoing efforts in the lab are exploring mechanisms to selectively disable tumor oxygen hyper-metabolism, as well as alternative approaches to further dampen hypoxia in cancer.

Learning Objectives:

1. Describe the impact of hypoxia on tumor immunity

2. Identify approaches for reducing tumor hypoxia

3. Describe the clinical impact of hypoxia reduction on sensitivity to CTLA-4 blockade

Gene editing experts within MilliporeSigma’s Cell Design Studio™ provide premier custom cell line engineering services to create and deliver unique cell-based assays tailored for drug discovery and preclinical manufacturing. Our cellular models have been used for applications such evaluating efficacy of CAR-T cells, screening candidate checkpoint inhibitors, and generating potency assays for therapeutic testing. We have also generated new functional biologically-relevant tumor-associated antigen and monoallelic HLA panel cell lines that express either individual tumor antigens at varying levels or individual HLAs. In this presentation, we will discuss the technology of these lines, their utility in immuno-oncology and novel therapeutic testing, and how they can fit in your workflow.

Learning Objectives:

1. Understand the limitations of cells commonly used in immuno-oncology assays

2. Learn about engineered tumor-associated antigen cell panels

3. Gain insight into the benefits of mono-allelic HLA expressing cell lines

Primary resistance to CD19-directed chimeric antigen receptor T-cell therapy (CART19) occurs in 10% to 20% of patients with acute lymphoblastic leukemia (ALL); however, the mechanisms of this resistance remain elusive. Using a genome-wide loss-of-function screen, we identified that impaired death receptor signaling in ALL led to rapidly progressive disease despite CART19 treatment. This was mediated by an inherent resistance to T-cell cytotoxicity that permitted antigen persistence and was subsequently magnified by the induction of CAR T-cell functional impairment. These findings were validated using samples from two CAR T-cell clinical trials in ALL, where we found that reduced expression of death receptor genes was associated with worse overall survival and reduced T-cell fitness. Our findings suggest that inherent dysregulation of death receptor signaling in ALL directly leads to CAR T-cell failure by impairing T-cell cytotoxicity and promoting progressive CAR T-cell dysfunction.

Inhibitory signals through the PD-1 pathway regulate T cell activation, tolerance, and exhaustion. To study the role of PD-1 in Treg cells, we generated mice that selectively lack PD-1 in T reg cells. PD-1–deficient Treg cells exhibit an activated phenotype and enhanced immunosuppressive function. The potent suppressive capacity of PD-1–deficient Treg is illustrated by protection from diabetes in NOD mice and ameliorated  EAE in mice lacking PD-1 selectively in Treg cells. Reduced PI3K–AKT pathway signaling is a mechanism underlying enhanced suppressive capacity of PD-1–deficient Treg. Our results show PD-1 is an “equal-opportunity” inhibitor of T cell activities. When PD-1 is lost on cells whose primary function is to suppress immune responses, the consequence is a stronger suppressor cell. This means that the outcome of PD-1 pathway blockade in vivo is a summation of increased Teffector and enhanced Treg suppressive activities.  The consequences for CTLA-4 combination therapy and for hyperprogression will be discussed. The frequency of PD-1+CD8+ T cells to PD-1+ Treg in the tumor microenvironment is a potential biomarker of PD-1 efficacy. Additional immune inhibitory pathways contribute to tumor immune evasion. HHLA2, a member of the B7 family of immunoregulatory ligands, has costimulatory effects through the CD28 family member TMIGD2, but HHLA2 also has inhibitory effects on T cells. We identified KIR3DL3 as an inhibitory receptor for HHLA2 in T and NK cells. We generated HHLA2 and KIR3DL3 antibodies that block the immune inhibitory activity of HHLA2 and enhance T cell and NK activities, preserving the costimulatory signal. HHLA2 is frequently expressed in several tumor types including clear cell renal cell carcinoma (ccRCC). HHLA2 expression was non-overlapping with PD-L1 expression, suggesting that HHLA2 mediates a mechanism of tumor immune evasion that is independent from PD-L1. Blockade of both PD-1 and KIR3DL3 pathways may be a more effective way to reverse tumor immune evasion.

Learning Objectives:

1. Understand how the outcome of PD-1 blockade is a summation of increased Teffector activity and increased Treg suppressive activity

2. Explain how PD-1 blockade leads to increased T reg activity

3. Understand a new checkpoint pathway, HHLA2-KIR3DL3

The Tumor Microenvironment (TME) is a highly complex niche, with many different cell types characterized by a range of biomarkers. Clever staining protocols like Cycle Immunofluorescence, and advanced imaging techniques such as Imaging Mass Cytometry can be used to probe these and other microenvironments in high detail. Although these techniques can acquire data in 30-100 channels, the analysis of these high-plex datasets still represents a considerable challenge.

During this presentation, Technical Specialist Dave Mason, will introduce the Visiopharm approach for the easy-to-use, automated, unbiased classification of high-plex data.

Learning Objectives:

1. The types of data you might encounter

2. Using machine learning and AI Deep Learning for robust cell detection - Easy multiplexing with the click of a button

3. Built-in plotting tools for exploratory analysis

Identifying reliable antibodies for use in immuno-oncology research is critical to address its recent growth and rapid expansion into clinical trials as mistakes in antibody validation impact patient health and wellbeing.  In this session, Dr. McWilliams discusses important validation considerations to make in your work to help ensure antibodies are on-target and performing as expected.  Unique challenges to antibody validation will be presented, providing a framework for why careful validation is needed and why collaboration between manufacturers and researchers is the best solution.  Recent publications and specific types of experiments will be explored to demonstrate how these experiments can best be applied to antibody validation and what to expect from the manufacturer of your antibodies.

Learning Objectives:

1. Understand the significance of antibody validation, including its needs and challenges

2. Gain insight into the antibody industry validation approach and learn steps to validate antibodies in your lab

3. How a collaborative approach to validation between researchers and manufacturers presents the best opportunity for long-term solutions

While providing indispensable insight into immune processes, the live cell imaging of immune cells poses unique challenges due to their non-adherent nature.  Suspension cells such as immune cells can easily move away from the FOV (field of view) during imaging by perturbation such as reagent introduction, often necessitating the use of surface coating to artificially immobilize cells.  In addition, unlike adherent cells, interacting suspension cells form aggregates, making it very difficult to track and analyze individual cells during imaging.  Here we describe the novel microfluidic-based dynamic live cell imaging platform CellASIC ONIX2, where microenvironmental parameters such as the perfusion of media/reagent, temperature, and gas compositions can be precisely controlled on-demand via software during the entire imaging experiment.  Unlike most live imaging platforms merely aiming to maintain temperature and gas at certain levels, CellASIC® ONIX2 platform’s ability to control fluidics and perfusion offers true on-demand control of cellular microenvironment during live cell imaging, and its standard plate form factor can instantly upgrade any existing inverted microscopes into a fully capable live cell imaging platform.  Overview of applications covering hypoxia, calcium imaging, apoptosis, migration, and suspension immune cell imaging will be presented.  Specific emphasis will be given to microfluidic designs targeted for use with different cell types as well as fluorescent probes for live cell imaging.  Any scientists planning to start live cell imaging experiments, as well as experienced imaging scientists wanting to broaden their applications will benefit from this webinar. 

Learning Objectives:

1. Understand the advantages and limitations of current live cell imaging platforms

2. Learn about the emerging field of microfluidic based dynamic live cell imaging

3. Learn various applications where dynamic live cell imaging can add value

This webinar will highlight the research solutions that Miltenyi Biotec has to offer for those investigating the roles of Tregs, NK cells, and Macrophages in the context of the tumor microenvironment.  We will discuss the different steps and considerations needed when working with these cells types, whether it is tumor processing, cell separation, cell culture or other downstream assays.

Learning Objectives:

1. Understand the importance and consequences of sample preparation

2. The fundamentals and advantages of MACS technology

3. Awareness of instruments, reagents and resources available from Miltenyi Biotec

Rhabdomyosarcoma (RMS) is a soft tissue cancer commonly arising in muscle and is the most common soft tissue cancer in children. Patients with metastatic RMS have a poor prognosis that has not improved in decades, highlighting the need for novel therapies. Pediatric tumors, and especially the fusion-gene driven alveolar RMS (aRMS) subset, have low mutational burden, suggesting that checkpoint therapies alone will be insufficient to control tumors. CAR-T cells are genetically engineered T lymphocytes expressing an extracellular binding domain and intracellular T cell signaling domains. Upon engagement of the binding domain to the antigenic target on a tumor cell, T cell-mediated cytotoxicity occurs. CAR specificity requires a molecular target that is tumor specific, expressed on the cell surface, and expressed at high enough levels for CAR-T activation. Previous work identified fibroblast growth factor receptor 4 (FGFR4, CD334) as specifically upregulated in RMS tumors compared to normal tissue, making it a candidate for selective targeting by CAR T cells. A previous CAR designed to target FGFR4 showed in vitro efficacy, and some control of tumor in a metastatic (intravenous) RMS model but failed to control orthotopic (intramuscular) RMS tumors in vivo. We have developed a new generation of FGFR4-binding moieties to be tested as new components of CARs. Our new binders were derived from both human F(ab)- and human VH-only phage display libraries. FGFR4 CARs were screened for surface expression by flow cytometry, cytotoxicity against target and non-target cells, and cytokine production in response to RMS cell lines in vitro. Two F(ab)-based FGFR4-binders were selected for further development against metastatic and intramuscular RMS tumor models in NSG mice. Additionally, we have modeled induced mechanisms of tumor defense in vitro and identified several potential mechanisms of tumor resistance to CAR T therapy which will be exploited to help armor engineered CAR-T cells to control RMS tumors. This data will determine whether our new FGFR4 CARs are sufficient for targeting and eliminating RMS tumors, provide insight into which armoring approaches are best suited to control RMS tumors, and thus develop a potent new therapeutic approach for rhabdomyosarcoma.

Learning Objectives:

1. Explain the disease conditions that make CAR T therapy a good therapeutic option

2. Understand rational CAR design and optimization

3. Describe tumor defense mechanisms and the corresponding CAR T armoring strategies

Prostate cancers are considered immunologically ‘cold’ tumors given the very few patients who respond to checkpoint inhibitor therapy (CPI). Recently, enrichment of interferon stimulated genes (ISGs) predicts a favorable response to CPI across various disease sites. The enhancer of zeste homolog-2 (EZH2) is over-expressed in prostate cancer and is known to negatively regulate ISGs. Here, we demonstrate that EZH2 inhibition in prostate cancer models activates a dsRNA-STING-ISG stress response upregulating genes involved in antigen presentation, Th-1 chemokine signaling, and interferon (IFN) response, including PD-L1 that is dependent on STING activation. EZH2 inhibition significantly increased intratumoral trafficking of activated CD8+ T-cells and increased M1 tumor associated macrophages (TAMs), overall reversing resistance to PD-1 CPI. Our study identifies EZH2 as a potent inhibitor of antitumor immunity and responsiveness to CPI. This data suggests EZH2 inhibition as a therapeutic direction to enhance prostate cancer response to PD-1 CPI.

Learning Objectives:

1. Identify epigenetic mechanisms important for prostate cancer immune evasion.

2. Define EZH2 inhibition has a rational approach to reverse prostate cancer resistance to check-point inhibitors.

Chimeric antigen receptors (CARs) are artificial T cell receptors that re-target patients’ T cells to specifically recognize and kill tumor cells. CAR T cell therapies targeting CD19 and now BCMA are FDA-approved and revolutionizing treatment of refractory B cell cancers. However, there are several limitations to current treatments and the application of CAR T cells to treat additional diseases, including toxicities and antigen-loss leading to relapse. To address these issues, we and others have developed “universal” CARs for which antigen-specificity is directed by one or more co-administered tumor-targeting antibodies. Universal CARs have several technical advantages over standard CAR T cell therapy including the ability to control activity by antibody dose, the ability to target multiple antigens on the same tumor to limit relapse, and the ability to be used across different cancer indications. Universal CAR T cells also come with unique technical challenges. In the seminar I will introduce universal CAR T cell design and mechanism of action, highlighting the universal “SNAP-CAR” technology developed in my lab and review other universal CARs in development. Finally, I will discuss technical challenges and future directions for universal CAR T cells including new mechanisms of conditional control potentially leading to greater tumor-targeting specificity.

Learning Objectives:

1. Define “universal CARs” and describe their mechanism of action.

2. Explain current challenges in CAR T cell therapy.

3. Describe potential benefits and challenges of therapy using T cells engineered with universal CARs.

Despite the promising results with current approved immunotherapies, all patients do not have the same level of response, and recent efforts have focused on understanding and avoiding immunotherapy resistance. Our lab and others have recently proposed that the metabolic state of tumor infiltrating lymphocytes, either preexisting or adoptively transferred, can compromise T cell function and consequently antitumor immunity. This suggests that the understanding of metabolic mediators and systemic metabolic disorders can contribute to the improvement of current immunotherapies as well as understanding therapeutic responses. Furthermore, understanding the mechanism of action in metabolic disorders can allow us to harness their inflammatory potential. Leptin affects a wide range of metabolic processes as well as being a pro inflammatory mediator, can be utilized as a means to remodel the metabolic state of the tumor microenvironment, consequently modulating immunotherapy response. Our data shows that leptin overexpressing tumors have increased T cell infiltration compared to control tumors, and these TIL are metabolically and functionally superior. We next wanted to translate tumor-specific expression of leptin into a therapeutic model. Oncolytic viruses are an attractive therapeutic modality promoting tumor specific killing, inducing an anti-tumor immune response, as well as the capacity to deliver genetic payloads potentially enhancing their anti-tumor activity. We designed an oncolytic Vaccinia virus that expresses leptin and used it to treat aggressive genetically-induced melanomas. While wild-type oncolytic Vaccinia showed some response, leptin-engineered Vaccinia had superior therapeutic efficacy inducing complete regressions in 30% of mice. TIL from these tumors have improved T cell infiltration and function. Single cell RNAseq and TCR sequencing immune infiltrates revealed the influx of new T cells by vaccinia while leptin-expressing virus showed specific clonal expansion. Most importantly we observed a strong memory response when complete responders where rechallenged with tumor cells. Taken together, these data suggest that understanding metabolic modulators in metabolic disorders, like leptin, can inform on therapeutic outcomes as well as taking advantage of these molecules for novel therapeutic modalities.

Learning Objectives:

1. Understand the role metabolic pressures play on the function of tumor infiltrating lymphocytes.

2. Identify metabolic mediators as potential therapeutic targets.

3. Learn about oncolytic viruses and their mechanisms of action as immunotherapeutic modalities.

IDO (indoleamine 2,3-dioxygenase) is an intracellular enzyme that regulates immune response by degrading tryptophan to kynurenine which results in a suppressive phenotype. IDO is over-expressed in multiple tumors including melanoma, and its higher expression is associate with worse prognosis. Multiple IDO inhibitors have been tested in clinical trials setting with different malignancies, majority of them are direct enzymatic inhibitors (epacadostat, linrodostat), as opposed to indoximod (a tryptophan memetic),  Acts directly on immune cells to reverse IDO pathway-mediated suppression. Preclinical data suggested the synergy between IDO and PD-1 inhibition along with multiple early phase clinical trials. In a phase 2 clinical trial combining indoximod and pembrolizumab in treatment naïve metastatic melanoma patients, the overall response rate (ORR) in 89 patients was 51% with confirmed complete response of 70% and median progression free survival of 12.4 months. The combination was well tolerated. With the negative phase 3 trial combining epacadostat and pembrolizumab in metastatic melanoma, the enthusiasm about IDO pathway is dampened, the reason for failure of this clinical trial is unknown. Questions have been raised about the reduced dose of epacadostat used (100 mg two times a day).  Multiple ongoing clinical trials currently evaluating different IDO inhibitors in different types of malignancies. Further evaluation is melanoma is still warranted.

Learning Objectives:

1. Learn about IDO pathway

2. IDO rule in cancer

Mechanisms of immune suppression in peripheral tissues counteract protective immunity to prevent immunopathology and are coopted by tumors for immune escape. While lymphatic vessels facilitate T cell priming through dendritic cell and antigen delivery to lymph nodes, they also exert PD-L1-dependent immune suppressive effects in lymph nodes at steady state, and facilitate melanoma metastasis. How lymphatic vessels may switch between immunogenic and immune suppressive roles remains and area of active investigation. Here we demonstrate a specific role for non-hematopoietic, non-tumor PD-L1, in limiting CD8+ T cell accumulation in melanoma. We find that IFNg produced by tissue infiltrating, antigen-specific CD8+ T cells is sufficient to activate lymphatic vessel PD-L1 expression. As such, disruption of IFNg-dependent crosstalk through lymphatic-specific loss of IFNγR boosts T cell accumulation in melanoma leading to CD8+ T cell-mediated tumor control and improved overall survival. Importantly, robust lymphatic vessel-dependent tumor control was revealed in the context of murine melanomas with significant UVB-induced somatic mutation burden, consistent with our model of cytotoxic T cell-activated mechanisms of negative feedback. Consequently, we present IFNg-mediated activation of tumor-associated lymphatic vessels as an important immunological switch in skin and tumors that contributes to adaptive immune resistance. We suggest that a continued understanding of context-dependent lymphatic vessel function will to reveal novel mechanisms of immune control and metastasis in melanoma.

Learning Objectives:

1. Define the impact of PD-L1 expressed on non-tumor, non-hematopoietic cells in T cell-mediated tumor control

2. Reveal the contribution of IFNg signaling in dermal lymphatic vessels to tumor control

3. Explain the contribution of adaptive immune resistance to immunotherapy response

Progress developing immune-based treatments to treat the highly lethal pancreatic ductal adenocarcinoma (PDA) has lagged behind other cancer types. This may be due, in part, to a fibroinflammatory and immunosuppressive tumor microenvironment (TME). To investigate mechanisms of resistance, we developed a novel mouse model to track pancreatic-tumor specific T cells.  While expression of the bioluminescent molecule click beetle red (CB) by tumor epithelial cells permits longitudinal imaging of pancreatic tumor growth in vivo, CB also and unexpectedly generates an immunodominant CD8 T cell epitope (CB101-109:H-2Db) in C57Bl/6 mice. Orthotopic implantation of CB+ PDA cells into syngeneic immunocompetent mice results in the activation, expansion and intratumoral accumulation of CB-specific T cells, thereby modeling the large fraction of PDA cases that have a significant T cell infiltrate. We created a peptide:MHC tetramer to detect CB-specific T cells and demonstrate robust T cell infiltrate. Nonetheless, PDA  induces a state of intratumoral T cell exhaustion characterized by loss of effector functions such as IFN-γ, TNF-α and GzmB and acquisition of regulatory functions such as the production of IL-10. We show that myeloid cells infiltrating PDA produce IL-27, which drives IL-10 production by T cells. Unexpectedly, αCD40 agonist ameliorates intratumoral myeloid cell IL-27 expression and T cell exhaustion, whereas αPD-L1 treatment promotes expansion of splenic CB-specific CD8 T cells. Combination agonistic αCD40 + αPD-L1 resulted in tumor clearance in 63% of animals and generated robust T cell memory. This therapy required IFNγ signaling whereas abrogating TNFα signaling resulted in cures in 100% of treated animals. In sum, this novel PDA animal model provides a tractable and rigorous longitudinal analysis of tumor-specific CD8 T cells which can inform the development of more effective immunotherapy combinations for patient treatment.

Learning Objectives:

1. Identify potential mechanisms driving resistance to immune therapy in pancreatic cancer

2. Appreciate the value of interrogating antigen-specific T cells in the context of disease

3. Elucidate how the tumor microenvironment plays a role in driving T cell exhaustion in malignancy and how combination therapies can operate to interfere with T cell exhaustion

Chimeric antigen receptor (CAR) T cells have demonstrated significant success in treating relapsed or treatment-refractory hematologic malignancies in the clinic, inducing remissions in 70-90% of pediatric patients with B-lineage acute lymphoblastic leukemia (B-ALL). Unfortunately, up to half of patients will eventually relapse, often due to poor CAR T cell expansion/persistence or modulation of the target antigen by tumor cells. One mechanism of antigen modulation is a decrease in surface expression of the CAR-targeted antigen, as seen in clinical trials with CAR T cells targeting the CD22 antigen in B-ALL, as well as in trials targeting solid tumors. Based on clinical data from our anti-CD22 CAR trial, we believe that sensitivity to low expression of the CD22 target antigen is important for efficacy of CARs targeting this antigen, and have shown in xengraft models that CAR T cells are less effective in responding to leukemia with low expression of the target antigen (CD22Lo).  Antigen downregulation thus represents a major mechanism of resistance to CAR T cell therapy and an area for improvement. Recent work from our lab and others have implicated the CAR antigen binding domain as an important factor in the efficacy of CARs responding to the CD22 target antigen. We have set out to characterize the effects of multiple modifications to the antigen binding domain on CAR responses to CD22Lo leukemia as a means to enhance overall CD22 CAR response rates. Collectively, these investigations will deepen our understanding of the factors important to designing CARs which are capable of targeting tumors with weakly-expressed antigens.

Learning Objectives:

1. Describe the impact of leukemic target antigen density on CAR T cells. 

2. Identify a way in which the CAR antigen binding domain can be modified. 

3. Identify one T cell function which is impacted by leukemic target antigen density. 

Ovarian cancer is a deadly disease which is usually diagnosed in advanced stages.  Our goal is to develop vaccines to prevent relapse.  Natural Th17 T cells protect against recurrence following conventional treatment.  Thus, we developed a Th17 inducing vaccine targeting the folate receptor alpha and immunized patients following first line therapy.  Vaccination resulted in prolonged relapse free survival and the development of immunity was associated with efficacy.  Findings from the clinical trial as well as early insights into mechanisms of action will be discussed along with a new phase II clinical trial evaluating efficacy in a randomized controlled setting.

Learning Objectives:

1. Describe basic principles of vaccination and tumor targets

2. Describe methods for generating Th17-inducing DC vaccines

3. Describe best uses and applications of vaccines in cancer patients

Immune checkpoint therapy (ICT) is a front-line treatment for lung cancer; however, low mutational burden and ‘non-T cell inflamed’ signatures predict poor responses to ICT in ~50% of patients. Adoptive cellular therapy (ACT) with T cells engineered to express T cell receptors (TCRs) specific for tumor-associated antigens (TAAs; proteins overexpressed by cancers) is an approach that circumvents the need for endogenous T cell responses. TCR-ACT is effective against hematologic cancers, but ACT against solid tumors is in the early stages of exploration. A deeper understanding of complex interactions between therapeutic cells and the tumor microenvironment (TME) is crucial for identifying successful strategies to enhance function and mitigate toxicity. Genetically engineered mouse models (GEMMs) achieve in situ tumor development alongside competent immune systems, recapitulating the native TME over the full spectrum of disease progression in a preclinical setting. Kras and p53 mutant (KP) GEMM lung tumors harbor few mutations, are poorly infiltrated by T cells, and are refractory to ICT, modeling intractable patient disease. We show that TAA-specific T cells recognize KP lung cancer cells and efficiently home to tumors, but rapidly lose function in the lung TME (compared to KP pancreatic tumors). Repetitive TCR-ACT can extend survival of animals with lung tumors, but animals eventually succumb to disease. These results show promise for TCR-ACT against lung cancer but highlight tissue-specific obstacles that must be overcome to enhance efficacy. We are continuing to leverage these models to advance our understanding of tissue-specific effects on therapeutic outcomes, and strategies to improve them. These findings offer inroads to uncovering more effective therapies, with the ultimate goal of benefitting cancer patients.

Learning Objectives: 

1. Define engineered T cell therapy and its potential utility in checkpoint inhibitor-recalcitrant tumor settings.

2. Understand the advantages (and disadvantages) of using genetically engineered preclinical models for studying tumor immunotherapy.

3. Learn about tissue specific factors that can contribute to suppression of anti-tumor T cell responses.