Radiation and anti-PD-L1 synergize by stimulating a stem-like T cell population in the tumor-draining lymph node.

Radiotherapy (RT) and anti-PD-L1 synergize to enhance local and distant (abscopal) tumor control. However, clinical results in humans have been variable. With the goal of improving clinical outcomes, we investigated the underlying synergistic mechanism focusing on a CD8+ PD-1+ Tcf-1+ stem-like T cell subset in the tumor-draining lymph node (TdLN). Using murine melanoma models, we found that RT + anti-PD-L1 induces a novel differentiation program in the TdLN stem-like population which leads to their expansion and differentiation into effector cells within the tumor. Our data indicate that optimal synergy between RT + anti-PD-L1 is dependent on the TdLN stem-like T cell population as either blockade of TdLN egress or specific stem-like T cell depletion reduced tumor control. Together, these data demonstrate a multistep stimulation of stem-like T cells following combination therapy which is initiated in the TdLN and completed in the tumor.

Engineering Improved CAR T Cell Products with A Multi-Cytokine Particle Platform for Hematologic and Solid Tumors.

Despite the remarkable clinical efficacy of chimeric antigen receptor (CAR) T cells in hematological malignancies, only a subset of patients achieves a durable complete response (dCR). DCR has been correlated with CAR T cell products enriched with T cells memory phenotypes. Therefore, reagents that consistently promote memory phenotypes during the manufacturing of CAR T cells have the potential to significantly improve clinical outcomes. A novel modular multi-cytokine particle (MCP) platform is developed that combines the signals necessary for activation, costimulation, and cytokine support into a single "all-in-one" stimulation reagent for CAR T cell manufacturing. This platform allows for the assembly and screening of compositionally diverse MCP libraries to identify formulations tailored to promote specific phenotypes with a high degree of flexibility. The approach is leveraged to identify unique MCP formulations that manufacture CAR T cell products from diffuse large B cell patients   with increased proportions of memory-like phenotypes MCP-manufactured CAR T cells demonstrate superior anti-tumor efficacy in mouse models of lymphoma and ovarian cancer through enhanced persistence. These findings serve as a proof-of-principle of the powerful utility of the MCP platform to identify "all-in-one" stimulation reagents that can improve the effectiveness of cell therapy products through optimal manufacturing.

Trans-vaccenic acid reprograms CD8+ T cells and anti-tumour immunity.

Diet-derived nutrients are inextricably linked to human physiology by providing energy and biosynthetic building blocks and by functioning as regulatory molecules. However, the mechanisms by which circulating nutrients in the human body influence specific physiological processes remain largely unknown. Here we use a blood nutrient compound library-based screening approach to demonstrate that dietary trans-vaccenic acid (TVA) directly promotes effector CD8+ T cell function and anti-tumour immunity in vivo. TVA is the predominant form of trans-fatty acids enriched in human milk, but the human body cannot produce TVA endogenously1. Circulating TVA in humans is mainly from ruminant-derived foods including beef, lamb and dairy products such as milk and butter2,3, but only around 19% or 12% of dietary TVA is converted to rumenic acid by humans or mice, respectively4,5. Mechanistically, TVA inactivates the cell-surface receptor GPR43, an immunomodulatory G protein-coupled receptor activated by its short-chain fatty acid ligands6-8. TVA thus antagonizes the short-chain fatty acid agonists of GPR43, leading to activation of the cAMP-PKA-CREB axis for enhanced CD8+ T cell function. These findings reveal that diet-derived TVA represents a mechanism for host-extrinsic reprogramming of CD8+ T cells as opposed to the intrahost gut microbiota-derived short-chain fatty acids. TVA thus has translational potential for the treatment of tumours.

Siglec-15 Promotes Evasion of Adaptive Immunity in B-cell Acute Lymphoblastic Leukemia.

Siglec-15 (Sig15) has been implicated as an immune checkpoint expressed in solid tumor-infiltrating macrophages and is being targeted in clinical trials with mAbs to normalize the tumor immune microenvironment and stimulate antitumor immunity. However, the role of Sig15 in hematologic malignancies remains undefined. Sig15 mRNA and protein expression levels in hematologic malignancies were determined from publicly available databases, cell lines, and primary patient samples. Human B-cell acute lymphoblastic leukemia (B-ALL) cell lines were used to identify signaling pathways involved in the regulation of Sig15 expression. Secreted/soluble Sig15 and cytokine levels were measured from the plasma of children with leukemia and healthy controls. Knockdown and knockout of Siglec15 in a murine model of B-ALL was used to evaluate the effect of leukemia-derived Sig15 on the immune response to leukemia. We observed pathologic overexpression of Sig15 in a variety of hematologic malignancies, including primary B-ALL samples. This overexpression was driven by NFκB activation, which also increased the surface localization of Sig15. Secreted/soluble Sig15 was found to circulate at elevated levels in the plasma of children with B-ALL and correlated with an immune-suppressive cytokine milieu. Genetic inhibition of Sig15 in murine B-ALL promoted clearance of the leukemia by the immune system and a marked reversal of the immune-privileged leukemia bone marrow niche, including expanded early effector CD8+ T cells and reduction of immunosuppressive cytokines. Thus, Sig15 is a novel, potent immunosuppressive molecule active in leukemia that may be targeted therapeutically to activate T lymphocytes against leukemia cells. Significance: We demonstrate that Sig15 is overexpressed in hematologic malignancies driven by NFκB, is required for immune evasion in a mouse model of leukemia, and, for the first time, that it circulates at high levels in the plasma of children with leukemia.

Chimeric Antigen Receptor (CAR) T Cell Therapy for Glioblastoma.

Genetic modification of T cells to express chimeric antigen receptors (CARs) has yielded remarkable clinical outcomes and initiated a novel era for cancer immunotherapy. The impressive clinical responses seen in hematologic malignancies have led to the investigation of CAR T cells in solid tumors but attaining similar results has been challenging to date. Glioblastoma (GBM) presents a particularly challenging malignancy for treatment and despite some progress in treatments over the past decade, prognosis remains poor for the vast majority of patients. However, recent data support the clinical efficacy and safety of CAR T cell therapy in GBM. In this review, common challenges associated with treating GBM will be discussed in addition to how CAR T cells can overcome such barriers. Additionally, emerging techniques of optimizing CAR T cell therapy for GBM will be emphasized, highlighting the prospective promise of cellular immunotherapy.

Multipurposing CARs: Same engine, different vehicles.

T cells genetically engineered to recognize and eliminate tumor cells through synthetic chimeric antigen receptors (CARs) have demonstrated remarkable clinical efficacy against B cell leukemia over the past decade. This therapy is a form of highly personalized medicine that involves genetically modifying a patient's T cells to recognize and kill cancer cells. With the FDA approval of 5 CAR T cell products, this approach has been validated as a powerful new drug in the therapeutic armamentarium against cancer. Researchers are now studying how to expand this technology beyond its use in conventional polyclonal αß T cells to address limitations to the current therapy in cancer and applications beyond it. Considering the specific characteristics of immune cell from diverse lineages, several preclinical and clinical studies are under way to assess the advantages of CAR-redirected function in these cells and apply the lessons learned from CAR T cell therapy in cancer to other diseases.

Supporting the Next Generation of Scientists to Lead Cancer Immunology Research.

Recent success in the use of immunotherapy for a broad range of cancers has propelled the field of cancer immunology to the forefront of cancer research. As more and more young investigators join the community of cancer immunologists, the Arthur L. Irving Family Foundation Cancer Immunology Symposium provided a platform to bring this expanding and vibrant community together and support the development of the future leaders in the field. This commentary outlines the lessons that emerged from the inaugural symposium highlighting the areas of scientific and career development that are essential for professional growth in the field of cancer immunology and beyond. Leading scientists and clinicians in the field provided their experience on the topics of scientific trajectory, career trajectory, publishing, fundraising, leadership, mentoring, and collaboration. Herein, we provide a conceptual and practical framework for career development to the broader scientific community.

Using Adoptive Cellular Therapy for Localized Protein Secretion.

ABSTRACT: Redirection of T cell cytotoxicity by the chimeric antigen receptor (CAR) structure may not be sufficient for optimal antitumor function in the patient tumor microenvironment. Comodifying CAR T cells to secrete different classes of proteins can be used to optimize CAR T cell function, overcome suppressive signals, and/or alter the tumor microenvironment milieu. These modifications aim to improve initial responses to therapy and enhance the durability of response. Furthermore, CAR T cells can deliver these molecules locally to the tumor microenvironment, avoiding systemic distribution. This approach has been tested in preclinical models using a variety of different classes of agonistic and antagonistic proteins, and clinical trials are currently underway to assess efficacy in patients.

Engineered Cytokines for Cancer and Autoimmune Disease Immunotherapy.

Cytokine signaling is critical to a range of biological processes including cell development, tissue repair, aging, and immunity. In addition to acting as key signal mediators of the immune system, cytokines can also serve as potent immunotherapies with more than 20 recombinant products currently Food and Drug Administration (FDA)-approved to treat conditions including hepatitis, multiple sclerosis, arthritis, and various cancers. Yet despite their biological importance and clinical utility, cytokine immunotherapies suffer from intrinsic challenges that limit their therapeutic potential including poor circulation, systemic toxicity, and low tissue- or cell-specificity. In the past decade in particular, methods have been devised to engineer cytokines in order to overcome such challenges and here, the myriad strategies are reviewed that may be employed in order to improve the therapeutic potential of cytokine and chemokine immunotherapies with applications in cancer and autoimmune disease therapy, as well as tissue engineering and regenerative medicine. For clarity, these strategies are collected and presented as they vary across size scales, ranging from single amino acid substitutions, to larger protein-polymer conjugates, nano/micrometer-scale particles, and macroscale implants. Together, this work aims to provide readers with a timely view of the field of cytokine engineering with an emphasis on early-stage therapeutic approaches.

Tumor derived UBR5 promotes ovarian cancer growth and metastasis through inducing immunosuppressive macrophages.

Immunosuppressive tumor microenvironment (TME) and ascites-derived spheroids in ovarian cancer (OC) facilitate tumor growth and progression, and also pose major obstacles for cancer therapy. The molecular pathways involved in the OC-TME interactions, how the crosstalk impinges on OC aggression and chemoresistance are not well-characterized. Here, we demonstrate that tumor-derived UBR5, an E3 ligase overexpressed in human OC associated with poor prognosis, is essential for OC progression principally by promoting tumor-associated macrophage recruitment and activation via key chemokines and cytokines. UBR5 is also required to sustain cell-intrinsic ß-catenin-mediated signaling to promote cellular adhesion/colonization and organoid formation by controlling the p53 protein level. OC-specific targeting of UBR5 strongly augments the survival benefit of conventional chemotherapy and immunotherapies. This work provides mechanistic insights into the novel oncogene-like functions of UBR5 in regulating the OC-TME crosstalk and suggests that UBR5 is a potential therapeutic target in OC treatment for modulating the TME and cancer stemness.

Optimization of T-cell Receptor-Modified T Cells for Cancer Therapy.

T-cell receptor (TCR)-modified T-cell gene therapy can target a variety of extracellular and intracellular tumor-associated antigens, yet has had little clinical success. A potential explanation for limited antitumor efficacy is a lack of T-cell activation in vivo We postulated that expression of proinflammatory cytokines in TCR-modified T cells would activate T cells and enhance antitumor efficacy. We demonstrate that expression of interleukin 18 (IL18) in tumor-directed TCR-modified T cells provides a superior proinflammatory signal than expression of interleukin 12 (IL12). Tumor-targeted T cells secreting IL18 promote persistent and functional effector T cells and a proinflammatory tumor microenvironment. Together, these effects augmented overall survival of mice in the pmel-1 syngeneic tumor model. When combined with sublethal irradiation, IL18-secreting pmel-1 T cells were able to eradicate tumors, whereas IL12-secreting pmel-1 T cells caused toxicity in mice through excessive cytokine secretion. In another xenograft tumor model, IL18 secretion enhanced the persistence and antitumor efficacy of NY-ESO-1-reactive TCR-modified human T cells as well as overall survival of tumor-bearing mice. These results demonstrate a rationale for optimizing the efficacy of TCR-modified T-cell cancer therapy through expression of IL18.See related commentary by Wijewarnasuriya et al., p. 732.

Excessive Costimulation Leads to Dysfunction of Adoptively Transferred T Cells.

Although clinical responses with CD19-targeting chimeric antigen receptor (CAR) T-cell treatment have been observed in patients with certain hematologic malignancies, high rates of disease relapse highlight the necessity to understand and improve mechanisms of CAR T-cell failure. Because T-cell dysfunction is thought to contribute to CAR T-cell treatment failure, understanding what mechanisms drive T cells into this dysfunctional state may aid optimal design of efficacious CAR T cells. Dysfunctional CAR T cells have been characterized as having upregulated inhibitory receptors and decreased cytolytic capabilities. Previous studies have identified a role for sustained CAR CD3ζ signaling in CAR T-cell dysfunction. Here, we demonstrate a mechanism that drives dysfunction in CAR T cells through excessive costimulation. Fully activated CD19-targeted CAR T cells were rendered dysfunctional upon stimulation with both endogenous CD28 stimulation and CAR-mediated CD28 costimulation. Costimulation-driven dysfunction of CAR T cells was demonstrated in a syngeneic immunocompetent mouse model, in which CAR T cells were activated with signals 1 (CD3ζ), 2 (CD28), and 3 (IL12). Thus, we show that CAR T-cell dysfunction can be driven through excessive CD28 and 4-1BB costimulation.See related article by Drakes et al., p. 743.

Engineering strategies to overcome the current roadblocks in CAR T cell therapy.

T cells genetically engineered to express chimeric antigen receptors (CARs) have proven - and impressive - therapeutic activity in patients with certain subtypes of B cell leukaemia or lymphoma, with promising efficacy also demonstrated in patients with multiple myeloma. Nevertheless, various barriers restrict the efficacy and/or prevent the widespread use of CAR T cell therapies in these patients as well as in those with other cancers, particularly solid tumours. Key challenges relating to CAR T cells include severe toxicities, restricted trafficking to, infiltration into and activation within tumours, suboptimal persistence in vivo, antigen escape and heterogeneity, and manufacturing issues. The evolution of CAR designs beyond the conventional structures will be necessary to address these limitations and to expand the use of CAR T cells to a wider range of malignancies. Investigators are addressing the current obstacles with a wide range of engineering strategies in order to improve the safety, efficacy and applicability of this therapeutic modality. In this Review, we discuss the innovative designs of novel CAR T cell products that are being developed to increase and expand the clinical benefits of these treatments in patients with diverse cancers.

Modeling anti-CD19 CAR T cell therapy in humanized mice with human immunity and autologous leukemia.

BACKGROUND: Adoptive immunotherapy using T cells expressing chimeric antigen receptors (CARs) targeting CD19 has produced remarkable clinical outcomes. However, much of the mechanisms of action, such as the development of memory responses and sources of immune cytokines, remain elusive largely due to the challenge of characterizing human CAR T cell function in vivo. The lack of a suitable in vivo model also hinders the development of new CAR T cell therapies. METHODS: We established a humanized mouse (hu-mouse) model with a functional human immune system and genetically-matched (autologous) primary acute B-lymphoblastic leukemia (B-ALL) that permits modeling of CD19-targeted CAR T cell therapy in immunocompetent hosts without allogeneic or xenogeneic immune responses. FINDINGS: Anti-CD19 CAR T cells were detected in blood of leukemic hu-mice with kinetics and levels similar to those seen in patients receiving CAR T cell therapy. The levels of CAR T cells were correlated inversely with the burden of leukemia cells and positively with the survival times in anti-CD19 CAR T cell-treated leukemic hu-mice. Infusion of anti-CD19 CAR T cells also resulted in rapid production of T cell- and monocyte/macrophage-derived cytokines and an increase in frequency of regulatory T cells as reported in clinical studies. INTERPRETATION: These results provide a proof-of-principle that this novel preclinical model has the potential to be used to model human CAR T cell therapy and facilitate the design of new CARs with improved antitumor activity.

Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo.

The efficacy of chimeric antigen receptor (CAR) T cell therapy against poorly responding tumors can be enhanced by administering the cells in combination with immune checkpoint blockade inhibitors. Alternatively, the CAR construct has been engineered to coexpress factors that boost CAR-T cell function in the tumor microenvironment. We modified CAR-T cells to secrete PD-1-blocking single-chain variable fragments (scFv). These scFv-secreting CAR-T cells acted in both a paracrine and autocrine manner to improve the anti-tumor activity of CAR-T cells and bystander tumor-specific T cells in clinically relevant syngeneic and xenogeneic mouse models of PD-L1+ hematologic and solid tumors. The efficacy was similar to or better than that achieved by combination therapy with CAR-T cells and a checkpoint inhibitor. This approach may improve safety, as the secreted scFvs remained localized to the tumor, protecting CAR-T cells from PD-1 inhibition, which could potentially avoid toxicities associated with systemic checkpoint inhibition.

Driving CAR T-cells forward.

The engineered expression of chimeric antigen receptors (CARs) on the surface of T cells enables the redirection of T-cell specificity. Early clinical trials using CAR T cells for the treatment of patients with cancer showed modest results, but the impressive outcomes of several trials of CD19-targeted CAR T cells in the treatment of patients with B-cell malignancies have generated an increased enthusiasm for this approach. Important lessons have been derived from clinical trials of CD19-specific CAR T cells, and ongoing clinical trials are testing CAR designs directed at novel targets involved in haematological and solid malignancies. In this Review, we discuss these trials and present strategies that can increase the antitumour efficacy and safety of CAR T-cell therapy. Given the fast-moving nature of this field, we only discuss studies with direct translational application currently or soon-to-be tested in the clinical setting.

CAR therapy for hematological cancers: can success seen in the treatment of B-cell acute lymphoblastic leukemia be applied to other hematological malignancies?

Chimeric antigen receptor (CAR) T-cell therapy has recently come into the spotlight due to impressive results in patients with B-cell acute lymphoblastic leukemia. By targeting CD19, a marker expressed most B-cell tumors, as well as normal B cells, CAR T-cell therapy has been investigated as a treatment strategy for B-cell leukemia and lymphoma. This review will discuss the successes of this therapy for the treatment of B-cell acute lymphoblastic leukemia and the challenges to this therapeutic strategy. We will also discuss application of CAR T-cell therapy to chronic lymphocytic leukemia and other B-cell malignancies including a follicular lymphoma, diffuse large B-cell lymphoma, as well as acute and plasma cell malignancies.

Ocaratuzumab, an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab's potency against CLL cells. In vitro assessment of ocaratuzumab's direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells. Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P<0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1-10 ug/ml; P<0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P<0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab. The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing countries.