Regina M. Young

Affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibit an Increased Therapeutic Index against Tumors in Mice

Target-mediated toxicity is a major limitation in the development of chimeric antigen T-cell receptors (CAR) for adoptive cell therapy of solid tumors. In this study, we developed a strategy to adjust the affinities of the scFv component of CAR to discriminate tumors overexpressing the target from normal tissues that express it at physiologic levels. A CAR-expressing T-cell panel was generated with target antigen affinities varying over three orders of magnitude. High-affinity cells recognized target expressed at any level, including at levels in normal cells that were undetectable by flow cytometry. Affinity-tuned cells exhibited robust antitumor efficacy similar to high-affinity cells, but spared normal cells expressing physiologic target levels. The use of affinity-tuned scFvs offers a strategy to empower wider use of CAR T cells against validated targets widely overexpressed on solid tumors, including those considered undruggable by this approach.

Hypoxia-mediated Selective mRNA Translation by an Internal Ribosome Entry Site-independent Mechanism

Although it is advantageous for hypoxic cells to inhibit protein synthesis and conserve energy, it is also important to translate mRNAs critical for adaptive responses to hypoxic stress. Because internal ribosome entry sites (IRES) have been postulated to mediate this preferential synthesis, we analyzed the 5 ′-untranslated regions from a panel of stress-regulated mRNAs for m7GTP cap-independent translation and identified putative IRES elements in encephalomyocarditis virus, vascular endothelial growth factor, hypoxia-inducible factors (HIFs) 1α and 2α, glucose transporter-like protein 1, p57Kip2, La, BiP, and triose phosphate isomerase transcripts. However, when capped and polyadenylated dicistronic RNAs were synthesized in vitro and transfected into cells, cellular IRES-mediated translation accounted for less than 1% that of the level of cap-dependent translation. Moreover, hypoxic stress failed to activate cap-independent synthesis, indicating that it is unlikely that this is the primary mechanism for the maintenance of the translation of these mRNAs under low O2. Furthermore, although HIF-1α is frequently cited as an example of an mRNA that is preferentially translated, we demonstrate that under different levels and durations of hypoxic stress, changes in newly synthesized HIF-1α and β-actin protein levels mirror alterations in corresponding mRNA abundance. In addition, our data suggest that cyclin-dependent kinase inhibitor p57Kip2 and vascular endothelial growth factor mRNAs are selectively translated by an IRES-independent mechanism under hypoxic stress.

Dysregulated mTORC1 renders cells critically dependent on desaturated lipids for survival under tumor-like stress

Solid tumors exhibit heterogeneous microenvironments, often characterized by limiting concentrations of oxygen (O2), glucose, and other nutrients. How oncogenic mutations alter stress response pathways, metabolism, and cell survival in the face of these challenges is incompletely understood. Here we report that constitutive mammalian target of rapamycin complex 1 (mTORC1) activity renders hypoxic cells dependent on exogenous desaturated lipids, as levels of de novo synthesized unsaturated fatty acids are reduced under low O2. Specifically, we demonstrate that hypoxic Tsc2(-/-) (tuberous sclerosis complex 2(-/-)) cells deprived of serum lipids exhibit a magnified unfolded protein response (UPR) but fail to appropriately expand their endoplasmic reticulum (ER), leading to inositol-requiring protein-1 (IRE1)-dependent cell death that can be reversed by the addition of unsaturated lipids. UPR activation and apoptosis were also detected in Tsc2-deficient kidney tumors. Importantly, we observed this phenotype in multiple human cancer cell lines and suggest that cells committed to unregulated growth within ischemic tumor microenvironments are unable to balance lipid and protein synthesis due to a critical limitation in desaturated lipids.

Effects of 4E-BP1 expression on hypoxic cell cycle inhibition and tumor cell proliferation and survival

Elevated activity of the eIF4F complex, which controls initiation of cap-dependent mRNA translation, has been linked to cancer progression. eIF4E recruitment to eIF4F is the rate limiting step of complex assembly and is regulated by eIF4E-Binding Proteins (4E-BPs). When stimulated, the mammalian Target of Rapamycin complex 1 (mTORC1) phosphorylates 4E-BP1, which then releases eIF4E. Hypoxia inhibits mTORC1 activity and therefore cap-dependent protein synthesis. To establish a novel genetic test of the role of eIF4F activity in regulating cell division and viability within hypoxic tumor microenvironments, we generated shRNA mediated 4E-BP1 knock-down in Rh30 rhabdomyosarcoma cells. 4E-BP1 knock-down relieved hypoxia-mediated inhibition of cycle progression in vitro and was correlated with increased expression of cyclin D1 and c-Myc. Xenograft tumors derived from these cells also displayed enhanced expression of cyclin D1 and c-Myc along with antiapoptotic genes encoding Bcl-xL, and XIAP, and failed to develop the extensive necrotic zones and edema observed in control tumors. Surprisingly, 4E-BP1 knock-down also leads to a dramatic increase in aberrant mitoses in vivo and enhanced expression of Mad2 and securin. Thus, reduced expression of the negative regulator of eIF4E has significant effects on tumor development, and is associated with enhanced cell proliferation and survival.

Physiological hypoxia promotes survival of cultured cortical neurons

Physiological oxygen (O2) tensions in brain tissues vary widely, from ≈ 5 to 40 Torr (1–6%), encompassing levels of moderate hypoxia that have often been considered neurotoxic in vitro. The effects of such hypoxia were examined in embryonic murine cortical neurons cultured continuously from plating in an atmosphere of 1% O2. Remarkably, cortical neurons thrived in 1% O2, with survival at 7–14 days significantly greater than that of neurons cultured in ambient conditions (20% O2). Immunostaining for microtubule‐associated protein‐2 (MAP‐2) and NeuN confirmed the neuronal identity of surviving cells, and demonstrated robust development of dendritic structures and MAP‐2 expression in hypoxia. Survival of neurons in 20% O2 could be promoted by transfer of medium conditioned by neurons in 1% O2, or by pharmacological induction of hypoxia‐inducible factor‐1α (HIF‐1α), suggesting a possible role for secreted factors under transcriptional regulation by HIF‐1 in the trophic effects of hypoxia. Vascular endothelial growth factor (VEGF), a factor regulated by HIF‐1, was strongly stimulated in neurons cultured in 1% O2. Treatment of neurons with exogenous VEGF partially improved survival in 20% O2, and inhibitors of VEGF action reduced survival of neurons in 1% O2. These data point to the dynamic role played by hypoxia, associated with HIF‐1 up‐regulation, in promoting survival of cortical neurons, in part through stimulation of VEGF expression and release.

Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia

Tolerance to self-antigens prevents the elimination of cancer by the immune system1,2. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45RO–CD8+ T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1–CD8+ CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies.An IL-6/STAT3 signature and memory CD8 T cell subset in preinfusion chimeric antigen receptor–expressing T cells associate with response in patients with high-risk chronic lymphocytic leukemia.

Augmentation of Antitumor Immunity by Human and Mouse CAR T Cells Secreting IL-18

SUMMARY The effects of transgenically encoded human and mouse IL-18 on T cell proliferation and its application in boosting chimeric antigen receptor (CAR) T cells are presented. Robust enhancement of proliferation of IL-18-secreting human T cells occurred in a xenograft model, and this was dependent on TCR and IL-18R signaling. IL-18 augmented IFN-γ secretion and proliferation of T cells activated by the endogenous TCR. TCR-deficient, human IL-18-expressing CD19 CAR T cells exhibited enhanced proliferation and antitumor activity in the xenograft model. Antigen-propelled activation of cytokine helper ensemble (APACHE) CAR T cells displayed inducible expression of IL-18 and enhanced antitumor immunity. In an intact mouse tumor model, CD19-IL-18 CAR T cells induced deeper B cell aplasia, significantly enhanced CAR T cell proliferation, and effectively augmented antitumor effects in mice with B16F10 melanoma. These findings point to a strategy to develop universal CAR T cells for patients with solid tumors.

Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation

Successful tumor eradication by chimeric antigen receptor-expressing (CAR-expressing) T lymphocytes depends on CAR T cell persistence and effector function. We hypothesized that CD4+ and CD8+ T cells may exhibit distinct persistence and effector phenotypes, depending on the identity of specific intracellular signaling domains (ICDs) used to generate the CAR. First, we demonstrate that the ICOS ICD dramatically enhanced the in vivo persistence of CAR-expressing CD4+ T cells that, in turn, increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB-based CARs. These data indicate that persistence of CD8+ T cells was highly dependent on a helper effect provided by the ICD used to redirect CD4+ T cells. Second, we discovered that combining ICOS and 4-1BB ICDs in a third-generation CAR displayed superior antitumor effects and increased persistence in vivo. Interestingly, we found that the membrane-proximal ICD displayed a dominant effect over the distal domain in third-generation CARs. The optimal antitumor and persistence benefits observed in third-generation ICOSBBz CAR T cells required the ICOS ICD to be positioned proximal to the cell membrane and linked to the ICOS transmembrane domain. Thus, CARs with ICOS and 4-1BB ICD demonstrate increased efficacy in solid tumor models over our current 4-1BB-based CAR and are promising therapeutics for clinical testing.

Safety and Efficacy of Intratumoral Injections of Chimeric Antigen Receptor (CAR) T Cells in Metastatic Breast Cancer

Transiently expressed chimeric antigen receptor T cells specific for c-Met, expressed in breast cancer, were injected into breast cancer tumors of six patients in a phase 0 clinical trial. Injections resulted in tumor necrosis and were well tolerated. Chimeric antigen receptors (CAR) are synthetic molecules that provide new specificities to T cells. Although successful in treatment of hematologic malignancies, CAR T cells are ineffective for solid tumors to date. We found that the cell-surface molecule c-Met was expressed in ∼50% of breast tumors, prompting the construction of a CAR T cell specific for c-Met, which halted tumor growth in immune-incompetent mice with tumor xenografts. We then evaluated the safety and feasibility of treating metastatic breast cancer with intratumoral administration of mRNA-transfected c-Met-CAR T cells in a phase 0 clinical trial (NCT01837602). Introducing the CAR construct via mRNA ensured safety by limiting the nontumor cell effects (on-target/off-tumor) of targeting c-Met. Patients with metastatic breast cancer with accessible cutaneous or lymph node metastases received a single intratumoral injection of 3 × 107 or 3 × 108 cells. CAR T mRNA was detectable in peripheral blood and in the injected tumor tissues after intratumoral injection in 2 and 4 patients, respectively. mRNA c-Met-CAR T cell injections were well tolerated, as none of the patients had study drug–related adverse effects greater than grade 1. Tumors treated with intratumoral injected mRNA c-Met-CAR T cells were excised and analyzed by immunohistochemistry, revealing extensive tumor necrosis at the injection site, cellular debris, loss of c-Met immunoreactivity, all surrounded by macrophages at the leading edges and within necrotic zones. We conclude that intratumoral injections of mRNA c-Met-CAR T cells are well tolerated and evoke an inflammatory response within tumors. Cancer Immunol Res; 5(12); 1152–61. ©2017 AACR.

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1–3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient’s second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient’s CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.Genetically engineered T cells that induced remission in a patient with chronic lymphocytic leukaemia were found to have disruption of the TET2 gene, which caused T cell changes that potentiated their anti-tumour effects.