Red Blood Cells Function as Reservoirs of Tumor DNA.

Novel screening techniques for early detection of lung cancer are urgently needed. Profiling circulating tumor cell-free DNA (ctDNA) has emerged as a promising tool for biopsy-free tumor genotyping. However, both the scarcity and short half-life of ctDNA substantially limit the sensitivity and clinical utility of ctDNA detection methodologies. Our discovery that Red Blood Cells (RBCs) sequester mitochondrial DNA opens a new avenue for detecting circulating nucleic acids, as RBCs represent an unrecognized reservoir of circulating nucleic acid. Here, we show that RBCs acquire tumor DNA following co-culture with lung cancer cell lines harboring KRAS and EGFR mutations. RBC-bound tumor DNA is detectable in patients with early-stage Non-Small Cell Lung Cancer (NSCLC) but not in healthy controls by qPCR. Our results collectively uncover a previously unrecognized yet easily accessible reservoir of tumor DNA, offering a promising foundation for future RBC-based tumor diagnostics.

IL7 increases targeted lipid nanoparticle-mediated mRNA expression in T cells in vitro and in vivo by enhancing T cell protein translation.

The use of lipid nanoparticles (LNP) to encapsulate and deliver mRNA has become an important therapeutic advance. In addition to vaccines, LNP-mRNA can be used in many other applications. For example, targeting the LNP with anti-CD5 antibodies (CD5/tLNP) can allow for efficient delivery of mRNA payloads to T cells to express protein. As the percentage of protein expressing T cells induced by an intravenous injection of CD5/tLNP is relatively low (4-20%), our goal was to find ways to increase mRNA-induced translation efficiency. We showed that T cell activation using an anti-CD3 antibody improved protein expression after CD5/tLNP transfection in vitro but not in vivo. T cell health and activation can be increased with cytokines, therefore, using mCherry mRNA as a reporter, we found that culturing either mouse or human T cells with the cytokine IL7 significantly improved protein expression of delivered mRNA in both CD4+ and CD8+ T cells in vitro. By pre-treating mice with systemic IL7 followed by tLNP administration, we observed significantly increased mCherry protein expression by T cells in vivo. Transcriptomic analysis of mouse T cells treated with IL7 in vitro revealed enhanced genomic pathways associated with protein translation. Improved translational ability was demonstrated by showing increased levels of protein expression after electroporation with mCherry mRNA in T cells cultured in the presence of IL7, but not with IL2 or IL15. These data show that IL7 selectively increases protein translation in T cells, and this property can be used to improve expression of tLNP-delivered mRNA in vivo.

Human Tumor-Associated Macrophages and Neutrophils Regulate Antitumor Antibody Efficacy through Lethal and Sublethal Trogocytosis.

The clinical benefits of tumor-targeting antibodies (tAb) are modest in solid human tumors. The efficacy of many tAbs is dependent on Fc receptor (FcR)-expressing leukocytes that bind Fc fragments of tAb. Tumor-associated macrophages (TAM) and neutrophils (TAN) represent the majority of FcR+ effectors in solid tumors. A better understanding of the mechanisms by which TAMs and TANs regulate tAb response could help improve the efficacy of cancer treatments. Here, we found that myeloid effectors interacting with tAb-opsonized lung cancer cells used antibody-dependent trogocytosis (ADT) but not antibody-dependent phagocytosis. During this process, myeloid cells "nibbled off" tumor cell fragments containing tAb/targeted antigen (tAg) complexes. ADT was only tumoricidal when the tumor cells expressed high levels of tAg and the effectors were present at high effector-to-tumor ratios. If either of these conditions were not met, which is typical for solid tumors, ADT was sublethal. Sublethal ADT, mainly mediated by CD32hiCD64hi TAM, led to two outcomes: (i) removal of surface tAg/tAb complexes from the tumor that facilitated tumor cell escape from the tumoricidal effects of tAb; and (ii) acquisition of bystander tAgs by TAM with subsequent cross-presentation and stimulation of tumor-specific T-cell responses. CD89hiCD32loCD64lo peripheral blood neutrophils (PBN) and TAN stimulated tumor cell growth in the presence of the IgG1 anti-EGFR Ab cetuximab; however, IgA anti-EGFR Abs triggered the tumoricidal activity of PBN and negated the stimulatory effect of TAN. Overall, this study provides insights into the mechanisms by which myeloid effectors mediate tumor cell killing or resistance during tAb therapy. SIGNIFICANCE: The elucidation of the conditions and mechanisms by which human FcR+ myeloid effectors mediate cancer cell resistance and killing during antibody treatment could help develop improved strategies for treating solid tumors.

CAR T cell therapy for patients with solid tumours: key lessons to learn and unlearn.

Chimeric antigen receptor (CAR) T cells have been approved for use in patients with B cell malignancies or relapsed and/or refractory multiple myeloma, yet efficacy against most solid tumours remains elusive. The limited imaging and biopsy data from clinical trials in this setting continues to hinder understanding, necessitating a reliance on imperfect preclinical models. In this Perspective, I re-evaluate current data and suggest potential pathways towards greater success, drawing lessons from the few successful trials testing CAR T cells in patients with solid tumours and the clinical experience with tumour-infiltrating lymphocytes. The most promising approaches include the use of pluripotent stem cells, co-targeting multiple mechanisms of immune evasion, employing multiple co-stimulatory domains, and CAR ligand-targeting vaccines. An alternative strategy focused on administering multiple doses of short-lived CAR T cells in an attempt to pre-empt exhaustion and maintain a functional effector pool should also be considered.

A genetically encoded protein tag for control and quantitative imaging of CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has been successful for hematological malignancies. Still, a lack of efficacy and potential toxicities have slowed its application for other indications. Furthermore, CAR T cells undergo dynamic expansion and contraction in vivo that cannot be easily predicted or controlled. Therefore, the safety and utility of such therapies could be enhanced by engineered mechanisms that engender reversible control and quantitative monitoring. Here, we use a genetic tag based on the enzyme Escherichia coli dihydrofolate reductase (eDHFR), and derivatives of trimethoprim (TMP) to modulate and monitor CAR expression and T cell activity. We fused eDHFR to the CAR C terminus, allowing regulation with TMP-based proteolysis-targeting chimeric small molecules (PROTACs). Fusion of eDHFR to the CAR does not interfere with cell signaling or its cytotoxic function, and the addition of TMP-based PROTACs results in a reversible and dose-dependent inhibition of CAR activity via the proteosome. We show the regulation of CAR expression in vivo and demonstrate imaging of the cells with TMP radiotracers. In vitro immunogenicity assays using primary human immune cells and overlapping peptide fragments of eDHFR showed no memory immune repertoire for eDHFR. Overall, this translationally-orientied approach allows for temporal monitoring and image-guided control of cell-based therapies.

Surgical Inflammation Alters Immune Response to Intraoperative Photodynamic Therapy.

Surgical cytoreduction for patients with malignant pleural mesothelioma (MPM) is used for selected patients as a part of multi-modality management strategy. Our group has previously described the clinical use of photodynamic therapy (PDT), a form of non-ionizing radiation, as an intraoperative therapy option for MPM. Although necessary for the removal of bulk disease, the effects of surgery on residual MPM burden are not understood. In this bedside-to-bench study, Photofrin-based PDT introduced the possibility of achieving a long-term response in murine models of MPM tumors that were surgically debulked by 60% to 90%. Thus, the addition of PDT provided curative potential after an incomplete resection. Despite this success, we postulated that surgical induction of inflammation may mitigate the comprehensive response of residual disease to further therapy. Utilizing a previously validated tumor incision (TI) model, we demonstrated that the introduction of surgical incisions had no effect on acute cytotoxicity by PDT. However, we found that surgically induced inflammation limited the generation of antitumor immunity by PDT. Compared with PDT alone, when TI preceded PDT of mouse tumors, splenocytes and/or CD8+ T cells from the treated mice transferred less antitumor immunity to recipient animals. These results demonstrate that addition of PDT to surgical cytoreduction significantly improves long-term response compared with cytoreduction alone, but at the same time, the inflammation induced by surgery may limit the antitumor immunity generated by PDT. These data inform future potential approaches aimed at blocking surgically induced immunosuppression that might improve the outcomes of intraoperative combined modality treatment. Significance: Although mesothelioma is difficult to treat, we have shown that combining surgery with a form of radiation, photodynamic therapy, may help people with mesothelioma live longer. In this study, we demonstrate in mice that this regimen could be further improved by addressing the inflammation induced as a by-product of surgery.

Desmoplastic stroma restricts T cell extravasation and mediates immune exclusion and immunosuppression in solid tumors.

The desmoplastic stroma in solid tumors presents a formidable challenge to immunotherapies that rely on endogenous or adoptively transferred T cells, however, the mechanisms are poorly understood. To define mechanisms involved, here we treat established desmoplastic pancreatic tumors with CAR T cells directed to fibroblast activation protein (FAP), an enzyme highly overexpressed on a subset of cancer-associated fibroblasts (CAFs). Depletion of FAP+ CAFs results in loss of the structural integrity of desmoplastic matrix. This renders these highly treatment-resistant cancers susceptible to subsequent treatment with a tumor antigen (mesothelin)-targeted CAR T cells and to anti-PD-1 antibody therapy. Mechanisms include overcoming stroma-dependent restriction of T cell extravasation and/or perivascular invasion, reversing immune exclusion, relieving T cell suppression, and altering the immune landscape by reducing myeloid cell accumulation and increasing endogenous CD8+ T cell and NK cell infiltration. These data provide strong rationale for combining tumor stroma- and malignant cell-targeted therapies to be tested in clinical trials.

Two cases of severe pulmonary toxicity from highly active mesothelin-directed CAR T cells.

Multiple clinical studies have treated mesothelin (MSLN)-positive solid tumors by administering MSLN-directed chimeric antigen receptor (CAR) T cells. Although these products are generally safe, efficacy is limited. Therefore, we generated and characterized a potent, fully human anti-MSLN CAR. In a phase 1 dose-escalation study of patients with solid tumors, we observed two cases of severe pulmonary toxicity following intravenous infusion of this product in the high-dose cohort (1-3 × 108 T cells per m2). Both patients demonstrated progressive hypoxemia within 48 h of infusion with clinical and laboratory findings consistent with cytokine release syndrome. One patient ultimately progressed to grade 5 respiratory failure. An autopsy revealed acute lung injury, extensive T cell infiltration, and accumulation of CAR T cells in the lungs. RNA and protein detection techniques confirmed low levels of MSLN expression by benign pulmonary epithelial cells in affected lung and lung samples obtained from other inflammatory or fibrotic conditions, indicating that pulmonary pneumocyte and not pleural expression of mesothelin may lead to dose-limiting toxicity. We suggest patient enrollment criteria and dosing regimens of MSLN-directed therapies consider the possibility of dynamic expression of mesothelin in benign lung with a special concern for patients with underlying inflammatory or fibrotic conditions.

Tissue-resident memory CAR T cells with stem-like characteristics display enhanced efficacy against solid and liquid tumors.

Chimeric antigen receptor (CAR) T cells demonstrate remarkable success in treating hematological malignancies, but their effectiveness in non-hematopoietic cancers remains limited. This study proposes enhancing CAR T cell function and localization in solid tumors by modifying the epigenome governing tissue-residency adaptation and early memory differentiation. We identify that a key factor in human tissue-resident memory CAR T cell (CAR-TRM) formation is activation in the presence of the pleotropic cytokine, transforming growth factor ß (TGF-ß), which enforces a core program of both "stemness" and sustained tissue residency by mediating chromatin remodeling and concurrent transcriptional changes. This approach leads to a practical and clinically actionable in vitro production method for engineering peripheral blood T cells into a large number of "stem-like" CAR-TRM cells resistant to tumor-associated dysfunction, possessing an enhanced ability to accumulate in situ and rapidly eliminate cancer cells for more effective immunotherapy.

Desmoplastic stroma restricts T cell extravasation and mediates immune exclusion and immunosuppression in solid tumors.

The desmoplastic stroma in solid tumors presents a formidable challenge to immunotherapies that rely on endogenous or adoptively transferred T cells, however, the mechanisms are poorly understood. To define mechanisms involved, we treat established desmoplastic pancreatic tumors with CAR T cells directed to fibroblast activation protein (FAP), an enzyme highly overexpressed on a subset of cancer-associated fibroblasts (CAFs). Depletion of FAP+CAFs results in loss of the structural integrity of desmoplastic matrix. This renders these highly treatment-resistant cancers susceptible to subsequent treatment with a tumor antigen (mesothelin)-targeted CAR and to anti-PD1 antibody therapy. Mechanisms include overcoming stroma-dependent restriction of T cell extravasation and/or perivascular invasion, reversing immune exclusion, relieving T cell suppression, and altering the immune landscape by reducing myeloid cell accumulation and increasing endogenous CD8+ T cell and NK cell infiltration. These data provide strong rationale for combining tumor stroma- and malignant cell-targeted therapies to be tested in clinical trials.

Na+/H+-exchanger 1 enhances antitumor activity of engineered NK-92 natural killer cells.

Adoptive cell transfer (ACT) immunotherapy has remarkable efficacy against some hematological malignancies. However, its efficacy in solid tumors is limited by the adverse tumor microenvironment (TME) conditions, most notably that acidity inhibits T and natural killer (NK) cell mTOR complex 1 (mTORC1) activity and impairs cytotoxicity. In several reported studies, systemic buffering of tumor acidity enhanced the efficacy of immune checkpoint inhibitors. Paradoxically, we found in a c-Myc-driven hepatocellular carcinoma model that systemic buffering increased tumor mTORC1 activity, negating inhibition of tumor growth by anti-PD1 treatment. Therefore, in this proof-of-concept study, we tested the metabolic engineering of immune effector cells to mitigate the inhibitory effect of tumor acidity while avoiding side effects associated with systemic buffering. We first overexpressed an activated RHEB in the human NK cell line NK-92, thereby rescuing acid-blunted mTORC1 activity and enhancing cytolytic activity. Then, to directly mitigate the effect of acidity, we ectopically expressed acid extruder proteins. Whereas ectopic expression of carbonic anhydrase IX (CA9) moderately increased mTORC1 activity, it did not enhance effector function. In contrast, overexpressing a constitutively active Na+/H+-exchanger 1 (NHE1; SLC9A1) in NK-92 did not elevate mTORC1 but enhanced degranulation, target engagement, in vitro cytotoxicity, and in vivo antitumor activity. Our findings suggest the feasibility of overcoming the inhibitory effect of the TME by metabolically engineering immune effector cells, which can enhance ACT for better efficacy against solid tumors.

Synthetic Secoisolariciresinol Diglucoside (LGM2605) Prevents Asbestos-Induced Inflammation and Genotoxic Cell Damage in Human Mesothelial Cells.

Although alveolar macrophages play a critical role in malignant transformation of mesothelial cells following asbestos exposure, inflammatory and oxidative processes continue to occur in the mesothelial cells lining the pleura that may contribute to the carcinogenic process. Malignant transformation of mesothelial cells following asbestos exposure occurs over several decades; however, amelioration of DNA damage, inflammation, and cell injury may impede the carcinogenic process. We have shown in an in vitro model of asbestos-induced macrophage activation that synthetic secoisolariciresinol diglucoside (LGM2605), given preventively, reduced inflammatory cascades and oxidative/nitrosative cell damage. Therefore, it was hypothesized that LGM2605 could also be effective in reducing asbestos-induced activation and the damage of pleural mesothelial cells. LGM2605 treatment (50 µM) of huma n pleural mesothelial cells was initiated 4 h prior to exposure to asbestos (crocidolite, 20 µg/cm2). Supernatant and cells were evaluated at 0, 2, 4, and 8 h post asbestos exposure for reactive oxygen species (ROS) generation, DNA damage (oxidized guanine), inflammasome activation (caspase-1 activity) and associated pro-inflammatory cytokine release (IL-1ß, IL-18, IL-6, TNFα, and HMGB1), and markers of oxidative stress (malondialdehyde (MDA) and 8-iso-prostaglandin F2a (8-iso-PGF2α). Asbestos induced a time-dependent ROS increase that was significantly (p < 0.0001) reduced (29.4%) by LGM2605 treatment. LGM2605 pretreatment also reduced levels of asbestos-induced DNA damage by 73.6% ± 1.0%. Although levels of inflammasome-activated cytokines, IL-1ß and IL-18, reached 29.2 pg/mL ± 0.7 pg/mL and 43.9 pg/mL ± 0.8 pg/mL, respectively, LGM2605 treatment significantly (p < 0.0001) reduced cytokine levels comparable to baseline (non-asbestos exposed) values (3.8 pg/mL ± 0.2 pg/mL and 5.4 pg/mL ± 0.2 pg/mL, respectively). Furthermore, levels of IL-6 and TNFα in asbestos-exposed mesothelial cells were high (289.1 pg/mL ± 2.9 pg/mL and 511.3 pg/mL ± 10.2 pg/mL, respectively), while remaining undetectable with LGM2605 pretreatment. HMGB1 (a key inflammatory mediator and initiator of malignant transformation) release was reduced 75.3% ± 0.4% by LGM2605. Levels of MDA and 8-iso-PGF2α, markers of oxidative cell injury, were significantly (p < 0.001) reduced by 80.5% ± 0.1% and 76.6% ± 0.3%, respectively. LGM2605, given preventively, reduced ROS generation, DNA damage, and inflammasome-activated cytokine release and key inflammatory mediators implicated in asbestos-induced malignant transformation of normal mesothelial cells.

Monitoring Therapeutic Response to Anti-FAP CAR T Cells Using [18F]AlF-FAPI-74.

PURPOSE: Despite the success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies, successful targeting of solid tumors with CAR T cells has been limited by a lack of durable responses and reports of toxicities. Our understanding of the limited therapeutic efficacy in solid tumors could be improved with quantitative tools that allow characterization of CAR T-targeted antigens in tumors and accurate monitoring of response. EXPERIMENTAL DESIGN: We used a radiolabeled FAP inhibitor (FAPI) [18F]AlF-FAPI-74 probe to complement ongoing efforts to develop and optimize FAP CAR T cells. The selectivity of the radiotracer for FAP was characterized in vitro, and its ability to monitor changes in FAP expression was evaluated using rodent models of lung cancer. RESULTS: [18F]AlF-FAPI-74 showed selective retention in FAP+ cells in vitro, with effective blocking of the uptake in presence of unlabeled FAPI. In vivo, [18F]AlF-FAPI-74 was able to detect FAP expression on tumor cells as well as FAP+ stromal cells in the tumor microenvironment with a high target-to-background ratio. We further demonstrated the utility of the tracer to monitor changes in FAP expression following FAP CAR T-cell therapy, and the PET imaging findings showed a robust correlation with ex vivo analyses. CONCLUSIONS: This noninvasive imaging approach to interrogate the tumor microenvironment represents an innovative pairing of a diagnostic PET probe with solid tumor CAR T-cell therapy and has the potential to serve as a predictive and pharmacodynamic response biomarker for FAP as well as other stroma-targeted therapies. A PET imaging approach targeting FAP expressed on activated fibroblasts of the tumor stroma has the potential to predict and monitor therapeutic response to FAP-targeted CAR T-cell therapy. See related commentary by Weber et al., p. 5241.

CAR T cells produced in vivo to treat cardiac injury.

Fibrosis affects millions of people with cardiac disease. We developed a therapeutic approach to generate transient antifibrotic chimeric antigen receptor (CAR) T cells in vivo by delivering modified messenger RNA (mRNA) in T cell­targeted lipid nanoparticles (LNPs). The efficacy of these in vivo­reprogrammed CAR T cells was evaluated by injecting CD5-targeted LNPs into a mouse model of heart failure. Efficient delivery of modified mRNA encoding the CAR to T lymphocytes was observed, which produced transient, effective CAR T cells in vivo. Antifibrotic CAR T cells exhibited trogocytosis and retained the target antigen as they accumulated in the spleen. Treatment with modified mRNA-targeted LNPs reduced fibrosis and restored cardiac function after injury. In vivo generation of CAR T cells may hold promise as a therapeutic platform to treat various diseases.

An NK-like CAR T cell transition in CAR T cell dysfunction.

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.

Serum soluble mesothelin-related protein (SMRP) and fibulin-3 levels correlate with baseline malignant pleural mesothelioma (MPM) tumor volumes but are not useful as biomarkers of response in an immunotherapy trial.

OBJECTIVES: Soluble mesothelin-related protein (SMRP) and fibulin-3 serum levels may serve as diagnostic and prognostic biomarkers of malignant pleural mesothelioma (MPM). Here, we evaluate these markers for correlation to tumor volume, prognosis and response assessment in a clinical trial of immunogene therapy in combination with chemotherapy. MATERIALS AND METHODS: Serial serum levels of SMRP and fibulin-3 were measured in adult patients with biopsy-proven MPM enrolled in two prospective clinical trials. Pre-therapy computed tomography (CT) measurements of tumor burden were calculated and correlated with pre-therapy serum SMRP and fibulin-3 levels in these two trials. Serological data were also correlated with radiological assessment of response using Modified RECIST criteria over the first 6 months of intrapleural delivery of adenovirus-IFN alpha (Ad.IFN-α) combined with chemotherapy. RESULTS: A cohort of 58 patients who enrolled in either a photodynamic therapy trial or immunotherapy clinical trial had available imaging and SMRP serological data for analysis of whom 45 patients had serological fibulin-3 data. The cohort mean total tumor volume was 387 cm3 (STD 561 cm3). Serum SMRP was detectable in 57 of 58 patients (mean 3.8 nM, STD 6.0). Serum fibulin-3 was detected in 44 of 45 patients (mean 23 ng/mL, STD 14). At pre-therapy baseline in these two trials, there was a strong correlation between tumor volume and serum SMRP levels (r = 0.61, p < 0.001), and a moderate correlation between tumor volume and serum fibulin-3 levels (r = 0.36, p = 0.014). Twenty-eight patients in the immunotherapy trial had longitudinal serologic and radiographic data. Fold-changes in SMRP and fibulin-3 did not show significant correlations with modified RECIST measurements. CONCLUSIONS: Although our data show correlations of SMRP and fibulin-3 with initial tumor volumes as measured by CT scanning, the use of SMRP and fibulin-3 as serological biomarkers in the immunotherapy trial were not useful in following tumor response longitudinally.

Mesothelin-targeted CAR-T cell therapy for solid tumors.

Introduction: Mesothelin (MSLN) is a tumor differentiation antigen normally restricted to the body's mesothelial surfaces, but significantly overexpressed in a broad range of solid tumors. For this reason, MSLN has emerged as an important target for the development of novel immunotherapies. This review focuses on anti-MSLN chimeric antigen receptor (CAR) T cell immunotherapy approaches.Areas covered: A brief overview of MSLN as a therapeutic target and existing anti-MSLN antibody-based drugs and vaccines is provided. A detailed account of anti-MSLN CAR-T cell approaches utilized in preclinical models is presented. Finally, a comprehensive summary of currently ongoing and completed anti-MSLN CAR-T cell clinical trials is discussed.Expert opinion: Initial trials using anti-MSLN CAR-T cells have been safe, but efficacy has been limited. Employing regional routes of delivery, introducing novel modifications leading to enhanced tumor infiltration and persistence, and improved safety profiles and combining anti-MSLN CAR-T cells with standard therapies, could render them more efficacious in the treatment of solid malignancies.

Neoadjuvant Gene-Mediated Cytotoxic Immunotherapy for Non-Small-Cell Lung Cancer: Safety and Immunologic Activity.

Gene-mediated cytotoxic immunotherapy (GMCI) is an immuno-oncology approach involving local delivery of a replication-deficient adenovirus expressing herpes simplex thymidine kinase (AdV-tk) followed by anti-herpetic prodrug activation that promotes immunogenic tumor cell death, antigen-presenting cell activation, and T cell stimulation. This phase I dose-escalation pilot trial assessed bronchoscopic delivery of AdV-tk in patients with suspected lung cancer who were candidates for surgery. A single intra-tumoral AdV-tk injection in three dose cohorts (maximum 1012 viral particles) was performed during diagnostic staging, followed by a 14-day course of the prodrug valacyclovir, and subsequent surgery 1 week later. Twelve patients participated after appropriate informed consent. Vector-related adverse events were minimal. Immune biomarkers were evaluated in tumor and blood before and after GMCI. Significantly increased infiltration of CD8+ T cells was found in resected tumors. Expression of activation, inhibitory, and proliferation markers, such as human leukocyte antigen (HLA)-DR, CD38, Ki67, PD-1, CD39, and CTLA-4, were significantly increased in both the tumor and peripheral CD8+ T cells. Thus, intratumoral AdV-tk injection into non-small-cell lung cancer (NSCLC) proved safe and feasible, and it effectively induced CD8+ T cell activation. These data provide a foundation for additional clinical trials of GMCI for lung cancer patients with potential benefit if combined with other immune therapies.

Gene signature of antigen processing and presentation machinery predicts response to checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma.

BACKGROUND: Limited data exist on the role of alterations in HLA Class I antigen processing and presentation machinery in mediating response to immune checkpoint blockade (ICB). METHODS: This retrospective cohort study analyzed transcriptional profiles from pre-treatment tumor samples of 51 chemotherapy-refractory advanced non-small cell lung cancer (NSCLC) patients and two independent melanoma cohorts treated with ICB. An antigen processing machinery (APM) score was generated utilizing eight genes associated with APM (B2M, CALR, NLRC5, PSMB9, PSME1, PSME3, RFX5, and HSP90AB1). Associations were made for therapeutic response, progression-free survival (PFS) and overall survival (OS). RESULTS: In NSCLC, the APM score was significantly higher in responders compared with non-responders (p=0.0001). An APM score above the median value for the cohort was associated with improved PFS (HR 0.34 (0.18 to 0.64), p=0.001) and OS (HR 0.44 (0.23 to 0.83), p=0.006). The APM score was correlated with an inflammation score based on the established T-cell-inflamed resistance gene expression profile (Pearson's r=0.58, p<0.0001). However, the APM score better predicted response to ICB relative to the inflammation score with area under a receiving operating characteristics curve of 0.84 and 0.70 for PFS and OS, respectively. In a cohort of 14 high-risk resectable stage III/IV melanoma patients treated with neoadjuvant anti-PD1 ICB, a higher APM score was associated with improved disease-free survival (HR: 0.08 (0.01 to 0.50), p=0.0065). In an additional independent melanoma cohort of 27 metastatic patients treated with ICB, a higher APM score was associated with improved OS (HR 0.29 (0.09 to 0.89), p=0.044). CONCLUSION: Our data demonstrate that defects in antigen presentation may be an important feature in predicting outcomes to ICB in both lung cancer and melanoma.

Current Advances in CAR T Cell Therapy for Malignant Mesothelioma.

Malignant mesothelioma is a relatively rare malignancy arising in the body's serosal surfaces, with malignant pleural mesothelioma (MPM) being the most common type. It is characterized by local spread within the thorax, poor prognosis and resistance to treatment. The development of various immunotherapeutic options has provided a new way- and hope- in treating cancer patients. Chimeric antigen receptor (CAR) T cell therapy has been proven very successful in treating hematological cancers, like leukemias and lymphomas, and its use is now being tested in solid tumors. CARs that recognize and bind to a specific tumor-associated antigen on the tumor's cell surface, are engineered and transduced into T cells. Interaction of the CAR T cell with the tumor then results in T cell activation and subsequent tumor cell lysis. In this review, we provide a current update on our previous comprehensive study summarizing the CAR T cell preclinical studies and clinical trials in MM, and discuss the future perspectives of CAR T cell therapy in this disease.