Cytotoxic Effector Function Research Articles

Targeting the EGF-Receptor and CD38 in Solid and Haematological Malignancies with Nanobody-Based Heavy Chain Antibodies and AAV Vectors

Abstract Background Although monoclonal antibodies binding the EGF-receptor (cetuximab, panitumumab) or CD38 (daratumumab) show therapeutic efficacy, many cancer patients still develop resistance. Therefore, new therapies are necessary. Nanobodies are highly soluble immunoglobulin domains derived from heavy chain antibodies (hcAbs) that naturally occur in camelids. Replication defective AAV (adeno associated virus) vectors, when equipped with a tumor targeting ligand, can be used for the delivery of (suicide) genes to tumor cells. Methods We generated recombinant chimeric nanobody-based human IgG1 hcAbs, by fusing EGFR- or CD38-specific nanobodies derived from immunized llamas to the hinge, CH2 and CH3 domains of human IgG1 (1, 2). To promote cytotoxic effector functions we introduced CDC (complement dependent cytotoxicity)-enhancing and ADCC (antibody-dependent cell-mediated cytotoxicity)-enhancing mutations into the CH2 and CH3 domains of human IgG1. As a second strategy to target CD38-overexpressing cells, we inserted CD38-specific nanobodies into the VP1 capsid protein of AAV2 (3). We analyzed the capacity of these constructs to target EGFR and CD38 in tumor cell lines and multiple myeloma (MM) patient bone marrow samples using proliferation, CDC, ADCC and AAV-transduction assays. Results EGFR-specific hcAbs effectively blocked the proliferation of tumor cell lines (HNSCC, head and neck squamous cell carcinoma and mCRC, metastatic colorectal cancer) expressing WT EGFR and cetuximab/panitumumab-escape variants of EGFR. EGFR- and CD38-specific hcAbs mediated effective ADCC towards tumor cell lines (HNSCC, mCRC and MM respectively). HcAbs carrying the hexabody mutation were more potent at inducing CDC than parental hcAbs. Display of CD38-specific nanobodies on the AAV capsid resulted in a 20- to 100-fold enhanced transduction of myeloma cells in patient bone marrow samples. Some of these results have recently been published (1, 2) or accepted for publication (3). We will present updated data on tumor cell lines and patient samples. Conclusion Nanobody-based hcAbs and nanobody-displaying AAV hold promise as novel tools to target solid and hematological tumors. Legal entity responsible for the study Friedrich Koch-Nolte. Funding DFG (German Research Foundation) and SFB (Collaborative Research Centres). Disclosure The author has declared no conflicts of interest.

Combining CAR Engineering and CIS Checkpoint Deletion in NK Cells for the Treatment of B Cell Hematologic Malignancies

Immune checkpoint-based therapies, which target the regulatory pathways of immunocompetent cells to enhance anti-tumor responses, have been at the heart of many recent clinical advances and have led to long-term remissions and possible cures. Most of this success was achieved with T-cells, but there are compelling reasons to predict that checkpoint ablation could modify NK cells in ways that would facilitate their antitumor activity. The suppressor of cytokine signaling (SOCS) family of proteins plays an important role in NK cell biology by attenuating cytokine signaling and effector function against cancer. One of its members, cytokine inducible SH2 containing protein (CIS), encoded by the CISH gene, is as an important checkpoint molecule in NK cells and is upregulated in response to IL-15. We hypothesized that CIS may act as a potent checkpoint in our iC9/CAR19/IL15 NK cells given the fact that they continuously produce IL-15, and that targeting this pathway would enhance their potency against B cell malignancies. In a series of in vitro studies, we showed that CISH is induced in iC9/CAR19/IL15 NK cells in a time dependent manner. To examine the functional consequences of CISH deletion in our CAR-NK cells, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing to silence CISH and retroviral transduction with the iC9/CAR19/IL15 construct. On day 7 we nucleofected the CAR transduced NK cells with Cas9 alone (Cas9 control) or Cas9 pre-loaded with crRNA:tracrRNA duplex targeting CISH exon 4. Gene editing efficiency was >90% as quantified by PCR and western blot. CISH knockout induced a phenotype characterized by the increased expression of markers of activation and cytotoxicity. These included granzyme-b, perforin, TRAIL and CD3z; transcription factors such as eomesodermin and T-bet; adaptor molecules such as DAP12; and activating coreceptors/proliferation markers such as DNAM, CD25 and Ki67. CISH knockout resulted in significantly enhanced function of iC9/CAR19/IL15 NK cells against Raji lymphoma evident by increased cytokine production (TNFa p=0.007, IFNg p=0.033) and degranulation (CD107a p=0.003) compared to Cas9 control cells. Moreover, CISH KO iC9/CAR.19-IL15 NK cells killed Raji lymphoma more efficiently than Cas9 control cells and formed a stronger immunologic synapse (p=0.037). RNA sequencing with gene set enrichment analysis (GSEA) confirmed enrichment of JAK/STAT signaling, TNFα and IFN-γ inflammatory response, mTORC1, and MYC hallmark pathways in CISH KO iC9/CAR19/IL15 NK cells compared to Cas9 control counterparts, providing a molecular mechanism for their enhanced effector function. Moreover, in an in-vivo NSG mouse model of Raji lymphoma, the antitumor activity of a single dose of CISH KO iC9/CAR19/IL15 transduced CB NK cells was significantly better than that of Cas9 control cells leading to a significant survival advantage (p=0.003) without evidence of increased toxicity. Thus, we demonstrate for the first time, that silencing a critical checkpoint in CAR-NK cells improves their potency, permitting greater cytotoxic effector function than seen with unmodified CAR-NK cells. Our data support the merging of CAR-engineering and immune checkpoint gene editing to enhance the therapeutic potential of NK cells. We are in the process of scaling up this approach in our GMP facility for translation to the clinic for the treatment of relapsed/refractory B cell hematologic malignancies. Disclosures Konopleva: Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding. Champlin:Sanofi-Genzyme: Research Funding; Actinium: Consultancy; Johnson and Johnson: Consultancy.

Affinity-matured ‘aquaporumab’ anti-aquaporin-4 antibody for therapy of seropositive neuromyelitis optica spectrum disorders

Pathogenesis in seropositive neuromyelitis optica spectrum disorders (herein called NMO) involves binding of IgG1 autoantibodies to aquaporin-4 (AQP4) on astrocytes in the central nervous system, which initiates complement and cellular injury. We previously developed an antibody blocking approach for potential therapy of NMO in which an engineered, monoclonal, anti-AQP4 antibody lacking cytotoxicity effector functions (called aquaporumab) blocked binding of NMO autoantibodies to astrocyte AQP4 (Tradtrantip et al. Ann. Neurol. 71, 314–322, 2012). Here, a high-affinity aquaporumab, which was generated by affinity maturation using saturation mutagenesis, was shown to block cellular injury caused by NMO patient sera. Anti-AQP4 antibody rAb-53, a fully human antibody with effector function neutralizing Fc mutations L234A/L235A and affinity-enhancing Fab mutations Y50R/S56R, called AQmabAM, bound to AQP4 in cell cultures with Kd ~ 18 ng/ml (~0.12 nM), ~8-fold greater affinity than the original antibody. AQmabAM, but without L234A/L235A Fc mutations, produced complement-dependent cytotoxicity (CDC) with EC50 ~ 82 ng/ml. AQmabAM prevented CDC produced by sera from eight NMO patients with IC50 ranging from 40 to 80 ng/ml, and similarly prevented antibody-dependent cellular cytotoxicity (ADCC). Mechanistic studies demonstrated that AQmabAM blocked binding of serum NMO autoantibodies to AQP4. AQmabAM offers a targeted, non-immunosuppressive approach for therapy of seropositive NMO. Autoantibody blocking may be a useful therapeutic strategy for other autoimmune diseases as well.

What Defines NK Cell Functional Fate: Phenotype or Metabolism?

NK cells are capable of an array of functions that range widely from their classic anti-tumor and anti-viral cytotoxic effector functions, to their critical regulatory roles in controlling inflammatory immune responses and promoting tissue growth. However, the mechanisms that polarize NK cells to these distinct and opposing functions are incompletely understood. NK cell functional subsets are primarily identified and studied based on phenotype, which has served as an accessible means for profiling NK cells and does offer information on NK cell activation state. However, inconsistencies have emerged in using classic phenotypes to inform function, which raise the questions: Can phenotype in fact define NK cell functional fate? What factors do profile and drive NK cell fate? In other immune cells, cell metabolism has been shown to critically determine subset polarization. There is a growing body of evidence that cell metabolism is integral to NK cell effector functions. Glucose-driven glycolysis and oxidative metabolism have been shown to drive classic NK cell anti-tumor and anti-viral effector functions. Recent studies have uncovered a critical role for metabolism in NK cell development, education, and memory generation. In this review, we will draw on the evidence to date to investigate the relationship between NK cell phenotype, metabolism, and functional fate. We explore a paradigm in which the differential activity of metabolic pathways within NK cells produce distinct metabolic fingerprints that comprehensively distinguish and drive the range of NK cell functional abilities. We will discuss future areas of study that are needed to develop and test this paradigm and suggest strategies to efficiently profile NK cells based on metabolism. Given the emerging role of metabolism in driving NK cell fates, profiling and modulating NK cell metabolism holds profound therapeutic potential to tune inflammatory and regulatory NK cell responses to treat disease.

Essential role of IκBNS for in vivo CD4+ T-cell activation, proliferation, and Th1-cell differentiation during Listeria monocytogenes infection in mice.

Acquisition of effector functions in T cells is guided by transcription factors, including NF‐κB, that itself is tightly controlled by inhibitory proteins. The atypical NF‐κB inhibitor, IκBNS, is involved in the development of Th1, Th17, and regulatory T (Treg) cells. However, it remained unclear to which extend IκBNS contributed to the acquisition of effector function in T cells specifically responding to a pathogen during in vivo infection. Tracking of adoptively transferred T cells in Listeria monocytogenes infected mice antigen‐specific activation of CD4+ T cells following in vivo pathogen encounter to strongly rely on IκBNS. While IκBNS was largely dispensable for the acquisition of cytotoxic effector function in CD8+ T cells, IκBNS‐deficient Th1 effector cells exhibited significantly reduced proliferation, marked changes in the pattern of activation marker expression, and reduced production of the Th1‐cell cytokines IFN‐γ, IL‐2, and TNF‐α. Complementary in vitro analyses using cells from novel reporter and inducible knockout mice revealed that IκBNS predominantly affects the early phase of Th1‐cell differentiation while its function in terminally differentiated cells appears to be negligible. Our data suggest IκBNS as a potential target to modulate specifically CD4+ T‐cell responses.

CXCR6+ NK Cells in Human Fetal Liver and Spleen Possess Unique Phenotypic and Functional Capabilities.

Tissue-resident Natural Killer (NK) cells vary in phenotype according to tissue origin, but are typically CD56bright, CXCR6+, and CD69+. NK cells appear very early in fetal development, but little is known about when markers of tissue residency appear during gestation and whether the expression of these markers, most notably the chemokine receptor CXCR6, are associated with differences in functional capability. Using multi-parametric flow cytometry, we interrogated fetal liver and spleen NK cells for the expression of a multitude of extracellular markers associated with NK cell maturation, differentiation, and migration. We analyzed total NK cells from fetal liver and spleen and compared them to their adult liver and spleen counterparts, and peripheral blood (PB) NK. We found that fetal NK cells resemble each other and their adult counterparts more than PB NK. Maturity markers including CD16, CD57, and KIR are lower in fetal NK cells than PB, and markers associated with an immature phenotype are higher in fetal liver and spleen NK cells (NKG2A, CD94, and CD27). However, T-bet/EOMES transcription factor profiles are similar amongst fetal and adult liver and spleen NK cells (T-bet−/EOMES+) but differ from PB NK cells (T-bet+EOMES−). Further, donor-matched fetal liver and spleen NK cells share similar patterns of expression for most markers as a function of gestational age. We also performed functional studies including degranulation, cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC) assays. Fetal liver and spleen NK cells displayed limited cytotoxic effector function in chromium release assays but produced copious amounts of TNFα and IFNγ, and degranulated efficiently in response to stimulation with PMA/ionomycin. Further, CXCR6+ NK cells in fetal liver and spleen produce more cytokines and degranulate more robustly than their CXCR6− counterparts, even though CXCR6+ NK cells in fetal liver and spleen possess an immature phenotype. Major differences between CXCR6− and + NK cell subsets appear to occur later in development, as a distinct CXCR6+ NK cell phenotype is much more clearly defined in PB. In conclusion, fetal liver and spleen NK cells share similar phenotypes, resemble their adult counterparts, and already possess a distinct CXCR6+ NK cell population with discrete functional capabilities.

The Potential for Cancer Immunotherapy in Targeting Surgery-Induced Natural Killer Cell Dysfunction.

Natural Killer (NK) cells are granular lymphocytes of the innate immune system that are able to recognize and kill tumor cells without undergoing clonal selection. Discovered over 40 years ago, they have since been recognized to possess both cytotoxic and cytokine-producing effector functions. Following trauma, NK cells are suppressed and their effector functions are impaired. This is especially important for cancer patients undergoing the removal of solid tumors, as surgery has shown to contribute to the development of metastasis and cancer recurrence postoperatively. We have recently shown that NK cells are critical mediators in the formation of metastasis after surgery. While research into the mechanism(s) responsible for NK cell dysfunction is ongoing, knowledge of these mechanisms will pave the way for perioperative therapeutics with the potential to improve cancer outcomes by reversing NK cell dysfunction. This review will discuss mechanisms of suppression in the postoperative environment, including hypercoagulability, suppressive soluble factors, the expansion of suppressive cell populations, and how this affects NK cell biology, including modulation of cell surface receptors, the potential for anergy, and immunosuppressive NK cell functions. This review will also outline potential immunotherapies to reverse postoperative NK dysfunction, with the goal of preventing surgery-induced metastasis.

Molecular Repolarisation of Tumour-Associated Macrophages.

The tumour microenvironment (TME) is composed of extracellular matrix and non-mutated cells supporting tumour growth and development. Tumour-associated macrophages (TAMs) are among the most abundant immune cells in the TME and are responsible for the onset of a smouldering inflammation. TAMs play a pivotal role in oncogenic processes as tumour proliferation, angiogenesis and metastasis, and they provide a barrier against the cytotoxic effector function of T lymphocytes and natural killer (NK) cells. However, TAMs are highly plastic cells that can adopt either pro- or anti-inflammatory roles in response to environmental cues. Consequently, TAMs represent an attractive target to recalibrate immune responses in the TME. Initial TAM-targeted strategies, such as macrophage depletion or disruption of TAM recruitment, have shown beneficial effects in preclinical models and clinical trials. Alternatively, reprogramming TAMs towards a proinflammatory and tumouricidal phenotype has become an attractive strategy in immunotherapy. This work summarises the molecular wheelwork of macrophage biology and presents an overview of molecular strategies to repolarise TAMs in immunotherapy.

Abstract B14: TCR transgenic T cells improve the anticancer potential of oncolytic vesicular stomatitis virus as cell carriers and as synergistic therapeutics

Abstract With the clinical approval of Talimogen laherparepvec (T-VEC), an oncolytic Herpes-simplex-Virus 1, in 2015, oncolytic viruses have gained much attention as a versatile and promising platform for cancer therapy. Vesicular stomatitis virus (VSV) represents an attractive oncolytic agent due to its inherent ability to preferentially infect and kill tumor cells, boost an antitumor immune response, and its flexibility to efficiently express transgenes. In order to overcome potential hurdles for the systemic administration of VSV, which include neutralization and nonspecific uptake, we aim to develop a combination therapy which employs T-cell receptor (TCR) transgenic T cells directed against an epitope of Myeloperoxidase (Klar et al. 2014, Leukemia). As genetically engineered T cells are currently undergoing clinical trials, they have the potential to provide an ideal vehicle for a combination therapy with VSV. In our work, we have shown that human CD8+ central memory T cells (CD8+ Tcm) not only transport and deliver infectious virus to their tumor target while eliciting their own potent cytotoxic effector functions, but they also support viral amplification in the absence of significantly decreased cell viability in culture after 24h of viral infection. Moreover, we demonstrate in coculture assays with ML2 leukemia cells, that TCR transgenic CD8+ Tcm profit from the additional virus-mediated tumor cell killing as compared to a monotherapy with TCR transgenic CD8+ Tcm alone. Although viral titers in coculture assays with TCR transgenic cells were slightly decreased compared to those achieved in cocultures with T cells not expressing the TCR, we consider this to be an additional benefit of the combination therapeutic approach to reduce VSV-mediated off-target side effects, as VSV is known for its neurotoxic and hepatotoxic effects in a dose dependent manner. This is supported by our findings that highly immune-deficient NSG mice tolerate similar amounts of virus better if applied together with CD8+ Tcm. Furthermore, we demonstrate that the combination therapy for tumor-bearing NSG mice leads to enhanced transduction efficiency of VSV compared to that achieved by delivery of naked virus, as well as faster tumor cell killing compared to monotherapies with TCR transgenic CD8+ T cm or VSV alone when applied at the maximum tolerated dose. We therefore conclude that the combination of these monotherapies is a crucial step toward a broader and safer option for cancer treatment, as each offers a complimentary approach to tumor cell killing while simultaneously reducing the weaknesses of the other treatment option. We speculate that immune evasion can be efficiently overcome by application of the combination therapy while providing an efficient targeting and delivery vehicle of systemically applied oncolytic VSV with a concomitant reduction of toxicity. As both, rVSV and transgenic T cells are already undergoing clinical testing, our proposed combination therapy has the potential for a seamless translation to clinical application. Citation Format: Michael Karl Melzer, Lisa Zeitlinger, Sabine Mall, Katja Steiger, Angela Krackhardt, Oliver Ebert, Jennifer Altomonte. TCR transgenic T cells improve the anticancer potential of oncolytic vesicular stomatitis virus as cell carriers and as synergistic therapeutics [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B14.

Blimp-1 induces and Hobit maintains the cytotoxic mediator granzyme B in CD8 Tcells.

CD8 Tcells acquire cytotoxic molecules including granzyme B during effector differentiation. Both tissue-resident memory CD8 Tcells (Trm) and circulating CD45RA+ effector-type Tcells (Temra) cells have the ability to retain granzyme B protein expression into the memory phase, but it is unclear how this persistence of cytolytic activity is regulated during steady state. Previously, we have described that the transcriptional regulators Hobit and Blimp-1 have overlapping target genes that include granzyme B, but their impact on the regulation of cytotoxicity in Trm and Temra cells during homeostasis has remained unclear. We examined the expression regulation of Hobit and Blimp-1 in murine and human CD8 T-cells to determine their timeframe of activity. While Blimp-1 mRNA was expressed throughout effector and memory Tcells, Blimp-1 protein, was only transiently expressed during the effector stage. In contrast, Hobit mRNA and protein expression was stably maintained during quiescence, but downregulated after activation. Notably, Blimp-1 was required for expression of granzyme B in murine effector Tcells and Trm, while Hobit specifically regulated granzyme B in murine Trm during the memory phase. These findings suggest that Blimp-1 initiates cytotoxic effector function and that Hobit maintains cytotoxicity in a deployment-ready modus in Trm.

Cytokine‐Functionalized Synthetic Dendritic Cells for T Cell Targeted Immunotherapies

AbstractWidespread application of cytokine‐based therapies is hampered by severe toxic side effects, resulting from off‐target (immune) cell stimulation. This emphasizes the need for more precise targeting of cytokines to immune cells. While cytokines are generally active as soluble proteins, it is demonstrated here that synthetic mimics of immune cells based on polyisocyanopeptides (PICs), called synthetic dendritic cells (sDCs) can efficiently present immobilized cytokines to T cells, when targeted by anti‐CD3 antibodies. Anti‐CD3/IL‐2‐functionalized PICs induce strong T cell activation and proliferation, when compared to PICs functionalized with interleukin‐2 (IL‐2) alone. In contrast to the semi‐flexible PICs, immobilization of IL‐2 on a rigid micro‐sized scaffold results in significant loss of IL‐2 activity, signifying the importance of molecular flexibility on the PIC polymers to maintain function. Similarly, anti‐CD3/IFNα‐functionalized PICs support long‐term proliferation of T cells. Interferon‐α containing sDCs additionally promote the development of cytotoxic effector functions of T cells with limited upregulation of the inhibitory immune checkpoint PD‐1. This high cytolytic activity and low PD‐1 expression are essential to maintain effective anti‐cancer activity. Together, these results demonstrate that PICs form unique nano‐sized scaffolds that efficiently present immobilized cytokines to T cells, thus creating a powerful tool to improve cytokine‐based immunotherapies without concomitant off‐target toxicity.

Abstract 5627: Anti-TIGIT biomarker study: Inhibition of TIGIT induces loss of Tregs from tumors and requires effector function for tumor growth inhibition

Abstract The immune checkpoint co-inhibitory receptor TIGIT (T cell immunoreceptor with Ig ITIM domain) is expressed on regulatory T cells (Tregs) and on activated CD4+ T, CD8+ T, and NK cells. We have reported that by blocking TIGIT activity with an IgG2a anti-TIGIT antibody (313R12), CD8+ and CD4+ Tcells and NK cells were activated, resulting in dose-dependent tumor growth inhibition (TGI) in multiple syngeneic mouse models. To explore the pharmacodynamics (PD) and mechanism of action of tumor growth inhibition (TGI) by anti-TIGIT antibodies, we examined the kinetics of immune cell frequency and activation in tumor by flow cytometry, qPCR and immunohistochemistry (IHC). We performed in vivo time course studies in the CT26.WT colon carcinoma model using weekly dosing at 0.1, 0.5 and 12.5 mg/kg anti-TGIT. Mice were sacrificed at 24 hours, 7 days and 14 days after the first dose for biomarker analysis. After 24 hours of treatment, Tregs in the tumor decreased and this reduction of Tregs was sustained at 7 and 14 days. Markers of immune cell activation and exhaustion such as CD69, PD1 and intracellular cytokines were modulated during the course of the study, suggesting a more cytotoxic intratumoral environment after 313R12 treatment. In addition, CD226, a binding partner of TIGIT, was significantly upregulated in T cells, Tregs and NK cells throughout the study, reflecting a feedback loop activated by inhibiting TIGIT activity. The anti-TIGIT antibody used in these studies, 313R12, is effector function competent and is able to induce cell-mediated cytotoxic effector functions upon binding. In order to determine whether effector function is necessary for anti-TIGIT antibody activity, we compared 313R12 with an effector function-deficient molecule, 313R13, in CT26.WT tumors. After 7 days, only 313R12 showed significant TGI compared to control-treated animals, suggesting that effector function is required for efficacy. While the effector function-deficient molecule 313R13 was able to similarly induce some changes in PD biomarkers, including immune cell activation, it required a higher dose than 313R12 to do so. To develop biomarkers for anti-TIGIT, we used microarray analyses to identify anti-TIGIT gene signatures in tumors and blood from multiple syngeneic models. In addition, we developed multiplexed IHC panels (e.g., TIGIT+CD8, TIGIT+FOXP3) to quantify expression of TIGIT and TIGIT ligand-positive immune cells in the tumor and surrounding stroma, and we profiled a panel of 80 human tumors with these panels. In summary, we examined the effects of anti-TIGIT antibodies on preclinical mouse models. Biomarker analysis demonstrated loss of Tregs and activation of T cells and NK cells, as well as effector function, as part of the mechanism of action of the molecule. We have also identified biomarkers that can be used for PD and potential predictive analysis in clinical trial samples. Citation Format: Gretchen M. Argast, Belinda Cancilla, Fiore Cattaruzza, Pete Yeung, Erwan le Scolan, Rose Harris, Reyhaneh Lahmy, Alayne Brunner, Min Wang, Gilbert O'Young, Earth Light Lowe, Fumiko Axelrod, Jorge Monteon, Jennifer Elechko, Andrew Lam, MingHong Xie, Austin Gurney, John Lewicki, Ann Kapoun. Anti-TIGIT biomarker study: Inhibition of TIGIT induces loss of Tregs from tumors and requires effector function for tumor growth inhibition [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 5627.

Defining the mechanisms responsible for the induction of cytotoxic CD4 T cell responses during viral infection

Abstract Induction of cytolytic lymphocytes is a core aspect of the anti-viral immune response. While NK and CD8 T cells are most often associated with the job of directly eliminating virus-infected cells, cytotoxic CD4 T cells can also be a major contributor. Cytotoxic CD4 T cell responses have been observed during a multitude of virus infections in both humans and mice including HIV, cytomegalovirus (CMV), herpes simplex virus, influenza virus, ectromelia virus (ECTV), and lymphocytic choriomeningitis virus. It has been previously demonstrated that the absence of a cytotoxic CD4 T cell response following ECTV infection of mice results in significantly greater viral burden. Runx3, the main transcription factor responsible for induction of cytolytic genes in NK and CD8 T cells, was also upregulated in cytotoxic CD4 T cells as compared to other activated or naive CD4 T cells following ECTV infection. Isolation of CD4 T cells expressing Runx3 revealed upregulated expression of cytolytic genes granzyme B and perforin. This suggests that Runx3 may play a critical role in the induction of cytotoxic effector functions in CD4 T cells. In addition, mice lacking expression of MHC class II on either CD11c+ or monocytic cells had significantly impaired cytotoxic CD4 T cell responses following ECTV infection. Challenge of mice with a mutant strain of ECTV that cannot spread from the footpad does not induce a cytotoxic CD4 T cell response, indicating that viremia or local replication is necessary. Our results highlight cell intrinsic and extrinsic mechanisms that are critical for the induction of the cytotoxic CD4 T cell response during viral infection.

Oncolytic viruses as engineering platforms for combination immunotherapy.

To effectively build on the recent successes of immune checkpoint blockade, adoptive T cell therapy and cancer vaccines, it is critical to rationally design combination strategies that will increase and extend efficacy to a larger proportion of patients. For example, the combination of anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and anti-programmed cell death protein 1 (PD1) immune checkpoint inhibitors essentially doubles the response rate in certain patients with metastatic melanoma. However, given the heterogeneity of cancer, it seems likely that even more complex combinations of immunomodulatory agents may be required to obtain consistent, durable therapeutic responses against a broad spectrum of cancers. This carries serious implications in terms of toxicities for patients, feasibility for care providers and costs for health-care systems. A compelling solution is offered by oncolytic viruses (OVs), which can be engineered to selectively replicate within and destroy tumour tissue while simultaneously augmenting antitumour immunity. In this Opinion article, we argue that the future of immunotherapy will include OVs that function as multiplexed immune-modulating platforms expressing factors such as immune checkpoint inhibitors, tumour antigens, cytokines and T cell engagers. We illustrate this concept by following the trials and tribulations of tumour-reactive T cells from their initial priming through to the execution of cytotoxic effector function in the tumour bed. We highlight the myriad opportunities for OVs to help overcome critical barriers in the T cell journey, leading to new synergistic mechanisms in the battle against cancer.

Turning the tables on cytomegalovirus: targeting viral Fc receptors by CARs containing mutated CH2\u2013CH3 IgG spacer domains

BackgroundDuring infection with human cytomegalovirus (HCMV) several viral proteins occur on cell surfaces in high quantity. We thus pursue an HLA-independent approach for immunotherapy of HCMV using chimeric antigen receptors (CARs) and bispecific BiTE® antibody constructs. In this context, HCMV-encoded proteins that mediate viral immune evasion and bind human IgG might represent particularly attractive target antigens. Unlike in observations of similar approaches for HIV and hepatitis B and C viruses, however, HCMV-infected cells develop a striking resistance to cytotoxic effector functions at later stages of the replication cycle. In our study we therefore wanted to test two hypotheses: (1) CAR T cells can efficiently inhibit HCMV replication independently from cytotoxic effector functions, and (2) HCMV can be targeted by CH2–CH3 IgG spacer domains that contain mutations previously reported to prevent exhaustion and to rescue CAR T cell function in vivo.MethodsReplication of GFP-encoding recombinant HCMV in fibroblasts in the presence and absence of supernatants from T cell co-cultures plus/minus cytokine neutralizing antibodies was analyzed by flow cytometry. CARs with wild type and mutated CH2–CH3 domains were expressed in human T cells by mRNA electroporation, and the function of the CARs was assessed by quantifying T cell cytokine secretion.ResultsWe confirm and extend previous evidence of antiviral cytokine effects and demonstrate that CAR T cells strongly block HCMV replication in fibroblasts mainly by combined secretion of IFN-γ and TNF. Furthermore, we show that fibroblasts infected with HCMV strains AD169 and Towne starting from day 3 have a high capacity for binding of human IgG1 and also strongly activate T cells expressing a CAR with CH2–CH3 domain. Importantly, we further show that mutations in the CH2–CH3 domain of IgG1 and IgG4, which were previously reported to rescue CAR T cell function by abrogating interaction with endogenous Fc receptors (FcRs), still enable recognition of FcRs encoded by HCMV.ConclusionsOur findings identify HCMV-encoded FcRs as an attractive additional target for HCMV immunotherapy by CARs and possibly bispecific antibodies. The use of specifically mutated IgG domains that bind to HCMV-FcRs without recognizing endogenous FcRs may supersede screening for novel binders directed against individual HCMV-FcRs.

Ex vivo-expanded NK cells from blood and ascites of ovarian cancer patients are cytotoxic against autologous primary ovarian cancer cells.

Ovarian cancer (OC) is the leading cause of gynecological cancer-related death in North America. Most ovarian cancer patients (OCPs) experience disease recurrence after first-line surgery and chemotherapy; thus, there is a need for novel second-line treatments to improve the prognosis of OC. Although peripheral blood-derived NK cells are known for their ability to spontaneously lyse tumour cells without prior sensitization, ascites-derived NK cells (ascites-NK cells) isolated from OCPs exhibit inhibitory phenotypes, impaired cytotoxicity and may play a pro-tumourigenic role in cancer progression. Therefore, it is of interest to improve the cytotoxic effector function of impaired OCP ascites-NK cells at the tumour environment. We investigated the efficacy of using an artificial APC-based ex vivo expansion technique to generate cytotoxic, expanded NK cells from previously impaired OCP ascites-NK cells, for use in an autologous model of NK cell immunotherapy. We are the first to obtain a log-scale expansion of OCP ascites-NK cells that upregulate the surface expression of activating receptors NKG2D, NKp30, NKp44, produce robust amounts of anti-tumour cytokines in the presence of OC cells and mediate direct tumour cytotoxicity against ascites-derived, primary OC cells obtained from autologous patients. Our findings demonstrate that it is possible to generate cytotoxic OCP ascites-NK cells from previously impaired OCP ascites-NK cells, which presents a promising immunotherapeutic target for the second-line treatment of OC. Future work should focus on evaluating the in vivo efficacy of autologous NK cell immunotherapy through the intraperitoneal delivery of NK cell expansion factors to a preclinical xenograft mouse model of human OC.

Deletion of TGF-β1 Increases Bacterial Clearance by Cytotoxic T Cells in a Tuberculosis Granuloma Model.

Mycobacterium tuberculosis is the pathogenic bacterium that causes tuberculosis (TB), one of the most lethal infectious diseases in the world. The only vaccine against TB is minimally protective, and multi-drug resistant TB necessitates new therapeutics to treat infection. Developing new therapies requires a better understanding of the complex host immune response to infection, including dissecting the processes leading to formation of granulomas, the dense cellular lesions associated with TB. In this work, we pair experimental and computational modeling studies to explore cytokine regulation in the context of TB. We use our next-generation hybrid multi-scale model of granuloma formation (GranSim) to capture molecular, cellular, and tissue scale dynamics of granuloma formation. We identify TGF-β1 as a major inhibitor of cytotoxic T-cell effector function in granulomas. Deletion of TGF-β1 from the system results in improved bacterial clearance and lesion sterilization. We also identify a novel dichotomous regulation of cytotoxic T cells and macrophages by TGF-β1 and IL-10, respectively. These findings suggest that increasing cytotoxic T-cell effector functions may increase bacterial clearance in granulomas and highlight potential new therapeutic targets for treating TB.

Essential Role of c-MYC for Natural Killer Cell Development, Proliferation and Anti-Tumor Activity

Natural killer (NK) cells are the major component of innate immunity with both cytotoxicity and cytokine producing effector functions. NK cells also regulate the interplay between innate immunity and adaptive immunity by secreting certain cytokines. Extrinsic regulators of NK cell development and function, including diverse ligands of NK cell receptors and cytokines from the microenvironment, have been extensively studied. However, intrinsic regulators for NK cell biology are still less understood. In our previous study on aggressive NK cell leukemia (ANKL), genomics and transcriptomics analyses indicated that c-MYC was universally upregulated and responsible for the proliferation and survival in ANKL cells (Manuscript in revision). Furthermore, STAT5, as a transcriptional regulator of c-MYC, was found to be essential in the survival and development of NK cells (Eckelhart et al., Blood 2011). In this regard, we want to understand the physiological and oncological roles of c-MYC in NK cells.To achieve our goal, we made two mouse models including c-Myc loss-of-function (LOF) and c-Myc gain-of-function (GOF) in NK cells. Ncr1Cre knock-in mice, in which Cre recombinase was inserted into the Nkp46 locus, was used. We crossed the c-Mycf/f mice with Ncr1Cre mice to generate the NK cell specific c-Myc LOF model c-MycΔ/Δ/Ncr1Cre. To generate the c-Myc GOF model, we crossed the Tg(tetO-MYC) mice with Ncr1Cre and Rosa26-Loxp-Stop-Loxp(LSL)-rtTA-GFP mice to get the Tg(tetO-MYC)/ Ncr1Cre/LSL-rtTA-GFP (iMYC) mice, in which c-Myc expression can be induced in a doxycycline dependent manner in NK cells. c-MycΔ/Δ/Ncr1Cre mice were analyzed between 6 to 14-weeks old. iMYC mice were induced by doxycycline from 6-weeks old for over 2 months and then analyzed. Wild type littermates were used as controls. In both models, mice were born normally and showed no obvious difference in growth compared to their littermates.In c-MycΔ/Δ/Ncr1Cre mice, a significant reduction of NK1.1+/DX5+ NK cell percentages in peripheral blood (3.6 ± 0.4% vs. 0.5 ± 0.1%, P < 0.0001, N=7) and spleen (2.4 ± 0.5% vs. 0.7 ± 0.1%, P < 0.01, N=6) was detected. In addition, the percentage of CD11b+ mature NK cells in the NK1.1+/DX5+ population was also reduced. In bone marrow (BM), although the total percentage of NK1.1+/DX5+ NK cells did not change, an obvious block of NK cell development was seen, as the majority of NK1.1+/DX5+ cells in BM were CD27+/CD11b- cells, which represent an immature pattern. To assess whether the NK cell proliferation is altered in this model, we performed BrdU labeling assays and found that BrdU incorporation rates decreased dramatically both in peripheral NK cells and BM NK progenitors. Functionally, we measured the IFN-γ secretion of splenic NK cells after PMA/Ionomycin stimulation. We found that the percentage of IFN-γ positive NK cells decreased significantly in c-MycΔ/Δ/Ncr1Cre mice (77.1 ± 7.0% vs. 53.8 ± 1.0%, N=3). Consistent with these data, the tumor surveillance was also severely impaired in this LOF model, as the number of lung metastatic sites significantly increased compared to the control mice in a B16F10 transplantation assay.In contrast to the LOF model, in our GOF model, the NK1.1+/DX5+ NK cell number in peripheral blood increased (3.1 ± 0.2% vs. 4.4 ± 0.5%, P < 0.05, N=7). Additionally, a small increase in the percentage of CD27-/CD11b+ population in NK1.1+/DX5+ cells was seen. Interestingly, however, the ability of IFN-γ secretion of splenic NK cells after PMA/Ionomycin stimulation was decreased in iMYC mice (72.8 ± 0.3% vs. 58.7 ± 1.1%, N=3), which showed the same alteration observed in c-MycΔ/Δ/Ncr1Cre mice. This result is probably not due to the impaired maturation, but rather it is the result of the higher percentage of CD27-/CD11b+ cells, which were considered terminally differentiated NK cells with lower cytotoxic functions.In summary, we found that c-Myc is essential for NK cell development, proliferation, and tumor surveillance. NK cell maturation and proliferation were impaired in the c-Myc LOF models and were boosted in the c-Myc GOF models. Our results also provide a mechanism basis for the potential application of targeting c-Myc in NK cells ex vivo or in vivo expansion, and NK-mediated immunotherapy. Future studies are needed to delineate the underlying mechanisms and explore the applications. DisclosuresNo relevant conflicts of interest to declare.

Cutting Edge: Murine NK Cells Degranulate and Retain Cytotoxic Function without Store-Operated Calcium Entry.

Sustained Ca2+ signaling, known as store-operated calcium entry (SOCE), occurs downstream of immunoreceptor engagement and is critical for cytotoxic lymphocyte signaling and effector function. CD8+ T cells require sustained Ca2+ signaling for inflammatory cytokine production and the killing of target cells; however, much less is known about its role in NK cells. In this study, we use mice deficient in stromal interacting molecules 1 and 2, which are required for SOCE, to examine the contribution of sustained Ca2+ signaling to murine NK cell function. Surprisingly, we found that, although SOCE is required for NK cell IFN-γ production in an NFAT-dependent manner, NK cell degranulation/cytotoxicity and tumor rejection in vivo remained intact in the absence of sustained Ca2+ signaling. Our data suggest that mouse NK cells use different signaling mechanisms for cytotoxicity compared with other cytotoxic lymphocytes.

Thy-1 stimulation of mouse T cells induces a delayed T cell receptor-like signal that results in Ca2+‑independent cytotoxicity.

Antibody-mediated crosslinking of Thy-1 [also known as cluster of differentiation (CD)90], results in a Tcell receptor (TcR)‑like signal; however, the impact of Thy‑1 stimulation in comparison to TcR stimulation on T cell activation and effector function has yet to be fully elucidated. In the present study, the outcome of Thy‑1‑and TcR‑induced stimulation of Tcells was investigated in mice, using fragment crystalizable(Fc) receptor‑bound antibodies and costimulatory signals provided by syngeneic lipopolysaccharide‑matured bone marrow‑derived dendritic cells. Compared with TcR signaling, Thy‑1 signaling initiated a less robust proliferative response in Tcells, as determined by tritiated‑thymidine incorporation. In addition, enzyme‑linked immunosorbent assays revealed that interleukin‑2 production was reduced, and the expression of CD25 and cyclinD3 was weaker in Thy‑1‑stimulated cells, as determined by western blotting; however, the expression of cyclin‑dependent kinase6 was similar to that in TcR‑induced Tcells. Furthermore, western blotting demonstrated that the phosphorylation of ζ-chain‑associated protein kinase70 and extracellular signal‑regulated kinase1/2 was delayed following Thy‑1 stimulation. DNA fragmentation assays revealed that cytotoxic effector function was also slower to develop in Thy‑1‑stimulated T cells, required more time to be effective and was largely Ca2+‑independent; these findings suggested that Fas ligand rather than granule‑associated perforin was involved in T cell effector function. In conclusion, the present results suggested that Thy‑1 signaling may contribute to the regulation of T cell homeostasis and the development of non‑specific T cell‑mediated cytotoxicity. However, further studies are required to elucidate the exact physiological roles of TcR‑like signals that result from Thy‑1 crosslinking and to investigate the molecular mechanisms that are involved.