Gina Scurti

TCR gene-modified T cells can efficiently treat established hepatitis C-associated hepatocellular carcinoma tumors

Abstract The success in recent clinical trials using T cell receptor (TCR)-genetically engineered T cells to treat melanoma has encouraged the use of this approach toward other malignancies and viral infections. Although hepatitis C virus (HCV) infection is being treated with a new set of successful direct anti-viral agents, potential for virologic breakthrough or relapse by immune escape variants remains. Additionally, many HCV+ patients have HCV-associated disease, including hepatocellular carcinoma (HCC), which does not respond to these novel drugs. Further exploration of other approaches to address HCV infection and its associated disease are highly warranted. Here, we demonstrate the therapeutic potential of PBL-derived T cells genetically engineered with a high-affinity, HLA-A2-restricted, HCV NS3:1406-1415-reactive TCR. HCV1406 TCR-transduced T cells can recognize naturally processed antigen and elicit CD8-independent recognition of both peptide-loaded targets and HCV+ human HCC cell lines. Furthermore, these cells can mediate regression of established HCV+ HCC in vivo. Our results suggest that HCV TCR-engineered antigen-reactive T cells may be a plausible immunotherapy option to treat HCV-associated malignancies, such as HCC.

Promoting Thiol Expression Increases the Durability of Antitumor T-cell Functions

Ex vivo-expanded CD8(+) T cells used for adoptive immunotherapy generally acquire an effector memory-like phenotype (TEM cells). With regard to therapeutic applications, two undesired features of this phenotype in vivo are limited persistence and reduced antitumor efficacy, relative to CD8(+) T cells with a central memory-like phenotype (TCM cells). Furthermore, there is incomplete knowledge about all the differences between TEM and TCM cells that may influence tumor treatment outcomes. Given that TCM cells survive relatively longer in oxidative tumor microenvironments, we investigated the hypothesis that TCM cells possess relatively greater antioxidative capacity than TEM cells. Here, we report that TCM cells exhibit a relative increase compared with TEM cells in the expression of cell surface thiols, a key target of cellular redox controls, along with other antioxidant molecules. Increased expression of redox regulators in TCM cells inversely correlated with the generation of reactive oxygen and nitrogen species, proliferative capacity, and glycolytic enzyme levels. Notably, T-cell receptor-transduced T cells pretreated with thiol donors, such as N-acetyl cysteine or rapamycin, upregulated thiol levels and antioxidant genes. A comparison of antitumor CD8(+) T-cell populations on the basis of surface thiol expression showed that thiol-high cells persisted longer in vivo and exerted superior tumor control. Our results suggest that higher levels of reduced cell surface thiols are a key characteristic of T cells that can control tumor growth and that profiling this biomarker may have benefits to adoptive T-cell immunotherapy protocols.

Hepatitis C virus‐cross‐reactive TCR gene‐modified T cells: a model for immunotherapy against diseases with genomic instability

A major obstacle hindering the development of effective immunity against viral infections, their associated disease, and certain cancers is their inherent genomic instability. Accumulation of mutations can alter processing and presentation of antigens recognized by antibodies and T cells that can lead to immune escape variants. Use of an agent that can intrinsically combat rapidly mutating viral or cancer‐associated antigens would be quite advantageous in developing effective immunity against such disease. We propose that T cells harboring cross‐reactive TCRs could serve as a therapeutic agent in these instances. With the use of hepatitis C virus, known for its genomic instability as a model for mutated antigen recognition, we demonstrate cross‐reactivity against immunogenic and mutagenic nonstructural protein 3:1406‐1415 and nonstructural protein 3:1073‐1081 epitopes in PBL‐derived, TCR‐gene‐modified T cells. These single TCR‐engineered T cells can CD8‐independently recognize naturally occurring and epidemiologically relevant mutant variants. TCR‐peptide MHC modeling data allow us to rationalize how TCR structural properties accommodate recognition of certain mutated epitopes and how these substitutions impact the requirement of CD8 affinity enhancement for recognition. A better understanding of such TCRs’ promiscuous behavior may allow for exploitation of these properties to develop novel, adoptive T cell‐based therapies for viral infections and cancers exhibiting similar genomic instability.

Efficacy of Adoptive T-cell Therapy Is Improved by Treatment with the Antioxidant N-Acetyl Cysteine, Which Limits Activation-Induced T-cell Death

Although adoptive transfer of autologous tumor antigen-specific T-cell immunotherapy can produce remarkable clinical efficacy, most patients do not achieve durable complete responses. We hypothesized that reducing susceptibility of T cells to activation-induced cell death (AICD), which increases during the rapid in vitro expansion of therapeutic T cells before their infusion, might improve the persistence of adoptively transferred cells. Our investigations revealed that repetitive stimulation of the T-cell receptor (TCR) induced AICD, as a result of activating the DNA damage response pathway through ATM-mediated Ser15 phosphorylation of p53. Activation of this DNA damage response pathway also occurred upon antigen-specific restimulation in TCR-transduced TIL1383I T cells prepared for adoptive transfer to patients as part of a clinical trial. Notably, treatment with the antioxidant N-acetyl cysteine (NAC) significantly reduced upregulation of the DNA damage marker γH2AX, subsequent ATM activation, and cell death. In the Pmel mouse model of melanoma, the presence of NAC during ex vivo T-cell expansion improved the persistence of adoptively transferred cells, reduced tumor growth, and increased survival. Taken together, our results offer a preclinical proof of concept for the addition of NAC to current therapeutic T-cell expansion protocols, offering immediate potential to improve the quality and therapeutic efficacy of adoptive T-cell therapeutics infused into patients. Cancer Res; 76(20); 6006-16. ©2016 AACR.

Enhanced stimulation of human tumor-specific T cells by dendritic cells matured in the presence of interferon-γ and multiple toll-like receptor agonists

Dendritic cell (DC) vaccines have been demonstrated to elicit immunological responses in numerous cancer immunotherapy trials. However, long-lasting clinical effects are infrequent. We therefore sought to establish a protocol to generate DC with greater immunostimulatory capacity. Immature DC were generated from healthy donor monocytes by culturing in the presence of IL-4 and GM-CSF and were further differentiated into mature DC by the addition of cocktails containing different cytokines and toll-like receptor (TLR) agonists. Overall, addition of IFNγ and the TLR7/8 agonist R848 during maturation was essential for the production of high levels of IL-12p70 which was further augmented by adding the TLR3 agonist poly I:C. In addition, the DC matured with IFNγ, R848, and poly I:C also induced upregulation of several other pro-inflammatory and Th1-skewing cytokines/chemokines, co-stimulatory receptors, and the chemokine receptor CCR7. For most cytokines and chemokines the production was even further potentiated by addition of the TLR4 agonist LPS. Concurrently, upregulation of the anti-inflammatory cytokine IL-10 was modest. Most importantly, DC matured with IFNγ, R848, and poly I:C had the ability to activate IFNγ production in allogeneic T cells and this was further enhanced by adding LPS to the cocktail. Furthermore, epitope-specific stimulation of TCR-transduced T cells by peptide- or whole tumor lysate-loaded DC was efficiently stimulated only by DC matured in the full maturation cocktail containing IFNγ and the three TLR ligands R848, poly I:C, and LPS. We suggest that this cocktail is used for future clinical trials of anti-cancer DC vaccines.

Critical biological parameters modulate affinity as a determinant of function in T-cell receptor gene-modified T-cells

T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR–pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR–pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR–pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR–pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.

Blocking TCR restimulation induced necroptosis in adoptively transferred T cells improves tumor control.

Advancements in adoptive cell transfer therapy (ACT) has led to the use of T cells engineered with tumor specific T cell receptors, which after rapid expansion can be obtained in sufficient numbers for treating patients. However, due to massive proliferation these cells are close to replicative senescence, exhibit exhausted phenotype, and also display increased susceptibility to activation induced cell death. We have previously shown that tumor reactive T cells undergo caspase-independent cell death upon TCR restimulation with cognate antigen, which involves reactive oxygen species and c-jun N-terminal kinase. Herein, we show that a large fraction of the human melanoma epitope tyrosinase reactive TCR transduced T cells that exhibit effector memory (TEM) phenotype and undergo programmed necrosis, or necroptosis, upon TCR restimulation. As compared to the T central memory (TCM) subsets, the TEM subset displayed an increased expression of genes involved in necroptotic cell death, and a necrotic phenotype upon TCR restimulation as confirmed by electron microscopy. Higher expression of receptor-interacting kinases (RIPK) that mediate necroptosis was also observed in the TEM fraction. Further, the TEM cells were rescued from undergoing necroptosis when pretreated with necroptotic inhibitor NecroX2 before TCR restimulation. Importantly, NecroX2 pretreated tumor reactive T cells also exhibited better tumor control and increased in vivo persistence when adoptively-transferred to treat subcutaneously established murine melanoma B16-F10. Thus, it is likely that the outcome of ACT could be vastly improved by interfering with the necroptotic cell death pathway in activated tumor reactive T cells used in immunotherapy.

Abstract B071: Enhanced IL-12 production and T cell stimulation ability by dendritic cells matured in presence of GMP-grade Toll-like receptor ligands and IFN-γ

There are numerous different DC maturation protocols but many contain non-GMP approved reagents and are thus not suitable for the production of vaccines to be used in patients. We aimed to generate an optimized DC product using GMP-grade reagents. Monocyte-derived DC were matured using a two-step maturation protocol. Monocytes were purified and cultured in CellGro® GMP DC medium for 48 hours in presence of IL-4 and GM-CSF plus 18 h with different combinations of TNF-α, IFN-γ and Toll-like receptor (TLR) agonists for TLR-3, -4 and -7/8. Following maturation, production of IL-12 was evaluated. Variations between individuals in the ability to respond to each maturation stimulus as well as in the overall IL-12 producing capacity was observed. The presence of both IFN-γgand TLR-7/8 agonist was essential for DC to produce high levels of IL-12. Furthermore, the highest induction of IL-12 production was consistently achieved by the combination of IFN-γ, TLR-3, -4 and -7/8 agonist. For TLR-4 triggering, synthetic monophosphoryl lipid A (MPLA) was used. Addition of non-GMP lipopolysaccharide (LPS) resulted in increased activation of DC. However, addition of MPLA did not result in any measurable activation of DC. Therefore, TLR-4 triggering was excluded. In absence of TLR-4 triggering, the combination including IFN-γ, TLR-3 and TLR-7/8 agonists induced the highest IL-12 levels. For TLR-7/8 triggering GMP-grade synthetic compound R848 was used throughout the study. For TLR-3 triggering poly I:C was used. Notably, poly I:C from different companies had high variability in the ability to activate DC. This was likely to a large extent due to contamination with LPS and possibly also other substances in non-GMP grade products. However, heterogeneity in the dsRNA molecule could also potentially affect the ability to trigger TLR-3 and cytosolic RNA-sensors. In the end we decided to use the GMP-grade, long poly I:C product Hiltonol®. The ability of the DC to activate allogeneic T cells to IFN-γ production was analyzed and correlated well to the production of IL-12 from the DC. This was also the case for tyrosinase-peptide loaded DC activation of tyrosinase-specific TCR transduced T cells. We are now investigating the ability of tumor-lysate loaded DC to trigger activation of tumor-specific T cells. In summary, we have developed a maturation protocol to generate GMP-grade DC with excellent ability to produce IL-12 and activate T cells. Citation Format: Lars Adamson, Dhifaf Sarhan, Bhavesh Choudhary, Jeroen Melief, Maria Nystrom, Ulrika Edback, Renee Vermeij, Gina Scurti, Michael Nishimura, Andreas Lundqvist, Rolf Kiessling, Tanja Lovgren. Enhanced IL-12 production and T cell stimulation ability by dendritic cells matured in presence of GMP-grade Toll-like receptor ligands and IFN-γ. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B071.