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M. Sadelain
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ORAL 28 - T Cell Therapy for Lung Cancer (ID 132)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 4
- Moderators:P.S. Adusumilli, E. Smit
- Coordinates: 9/08/2015, 16:45 - 18:15, Four Seasons Ballroom F1+F2
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ORAL28.02 - Mesothelin-Targeted CAR T-Cell Therapy for the Treatment of Heterogeneous Antigen-Expressing Lung Adenocarcinoma (ID 3172)
16:56 - 17:07 | Author(s): M. Sadelain
- Abstract
Background:
Adoptive T-cell therapy using chimeric antigen receptors (CAR) is an emerging strategy by redirecting T-cell effector functions against a cancer cell-surface antigen. To target lung adenocarcinoma (ADC) by CAR T-cell therapy, our laboratory has identified mesothelin (MSLN), a cell-surface antigen based on our published observation that MSLN is expressed in 60% of primary and metastatic lung ADC and is associated with tumor aggressiveness. Unlike hematological malignancies where CAR T-cell therapy has been successful targeting CD19, a cell-surface antigen that is uniformly expressed on B cells, MSLN expression intensity and distribution among lung ADC tumors is heterogeneous. The efficacy of CAR T-cell therapy in a heterogeneous antigen microenvironment is unknown. We hypothesized that the MSLN-targeted CAR T cells will be effective against high-antigen expressing lung ADC cells and the presence of even a small proportion of high MSLN expressing cells can enhance CAR T-cell cytotoxicity against low-antigen expressing lung ADC cells.
Methods:
Human peripheral blood T cells were retrovirally transduced with a 2[nd] generation of CAR targeting MSLN and bearing CD28 and CD3zeta activation domains. In vitro, we analyzed CAR T-cell cytotoxicity ([51]Cr release assay), effector cytokine secretion (Luminex assay), and proliferation (cell-counting assay) against lung ADC cell lines expressing variable levels of MSLN. In vivo, antitumor efficacy was evaluated by median survival and tumor bioluminescence (BLI) in mice bearing established homogeneous or heterogeneous lung ADC tumors.
Results:
In in vitro assays utilizing lung ADC cells with variable level of MSLN expression [low-antigen expression (EKVX or A549) or high-antigen expression (A549M and H1299M), control lung fibroblast (MRC5) or mesothelial cells (MET5A)], CAR T cells exhibit antigen-specific cytolytic activity, effector cytokine secretion and proliferation in proportion to the MSLN expression on cancer cells. In vivo, a single low dose of CAR T cells eradicates primary and metastatic established tumor expressing high-level of MSLN and prolongs tumor free survival (41 days vs not reached, p<0.0001). We next evaluated CAR T-cell efficacy in heterogeneous antigen microenvironment by mixing low and high antigen-expressing cells (A549 expressing firefly luciferase/A549M) and assessed the A549 tumor burden only by bioluminescence imaging. In the presence of A549M cells, CAR T cells are able to prolong progression-free survival of A549 tumor burden (22 days vs 0 days in absence of A549M cells). Further mechanistic studies demonstrated that CAR T cells lysed an additional 5%-15% A549 or EKVX cells in the presence of H1299M or A549M cells (p<0.05) without off-target cytotoxicity. Antigen-activated CAR T cells were effective against low-antigen expressing lung ADC cells without the need for high-antigen expressing cells in the coculture.
Conclusion:
Our results provide scientific rationale to translate MSLN-targeted CAR T-cell therapy for the treatment of the primary and metastatic lung ADC. A phase I clinical trial (NCT02414269) that includes lung ADC patients is initiated at our center.
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ORAL28.03 - Genetic-Engineering Strategies to Enhance CAR T-Cell Therapy Efficacy against PD-L1 Expressing Lung Adenocarcinoma and Mesothelioma (ID 3139)
17:07 - 17:18 | Author(s): M. Sadelain
- Abstract
Background:
This abstract is under embargo until September 8, 2015 and will be distributed onsite on September 8 in a Late Breaking Abstract Supplement.
Methods:
Results:
Conclusion:
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ORAL28.04 - Tumor-Targeted Radiation Therapy Helps Overcome the Solid Tumor T-Cell Infiltration Barrier and Promotes Mesothelin CAR T-Cell Therapy (ID 3142)
17:18 - 17:29 | Author(s): M. Sadelain
- Abstract
Background:
Translating recent chimeric antigen receptor (CAR) T-cell therapy successes in hematologic malignancies to solid cancers requires overcoming barriers unique to solid tumors such as inadequate tumor infiltration, proliferation, and persistence. Our laboratory has published the rationale to target mesothelin (MSLN), a cell-surface antigen expressed in the majority of thoracic malignancies. We hypothesized that the immune modulating effects of low-dose radiation therapy (RT) would enhance the infiltration and proliferation of mesothelin-targeted CAR T-cell therapy for thoracic cancers, thereby achieving long-term tumor eradication.
Methods:
Using human T cells retrovirally transduced to express mesothelin-targeted CARs, we evaluated T-cell cytotoxicity by chromium release assay, proliferation by cell count assay, cytokine-release by multiplex ELISA, phenotype by flow cytometry, and chemokine receptor profiles by PCR against MSLN-expressing mesothelioma and lung cancer cell lines with and without localized RT. In clinically relevant mouse models (NOD/SCID gamma mice) with established MSLN-expressing tumors, we monitored therapy response, T-cell kinetics and anti-tumor efficacy by utilizing bioluminescent imaging (BLI), and conducted flow cytometric analysis of splenic/peripheral blood T cells for characterization of CAR T-cell effector phenotype.
Results:
RT did not enhance CAR T-cell cytotoxicity. In vitro, RT enhanced CAR T-cell migration in chemotactic assays, and correlatively induced the secretion of chemokines by tumor cells (Fig.1A). In vivo, RT resulted in dose dependent chemokine secretion with robust early intratumoral CAR T-cell accumulation (p<0.05, Fig.1B) as demonstrated by T-cell BLI. Ex vivo tumor analysis by flow cytometry on day 7 post T-cell administration confirmed that RT increased early infiltration and proliferation (p<0.05). Also, single low-dose RT potentiated the efficacy of systemically administered CAR T cells (median survival 30d vs. 79d, p= 0.02) with at least 50% tumor eradication up to 100 days even with a 30-fold decreased dose (Fig.1C&D). Furthermore, in mice with tumor eradication, harvested spleen T-cell analysis at day 56 demonstrated a greater number of persisting CAR T cells in mice treated with RT (p=0.02, Fig.1E).Figure 1
Conclusion:
Our data provides the rationale to use localized RT as a preconditioning regimen prior to CAR T-cell administration in a clinical trial for thoracic malignancies. Furthermore, our mechanistic observation of RT-induced, chemokine-mediated, enhanced T-cell infiltration may also assist the trafficking of endogenous anti-tumor T cells, thereby shifting the balance towards a cohesive anti-tumor immune microenvironment.
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ORAL28.05 - Mesothelin and MUC16 (CA125) Are Antigen-Targets for CAR T-Cell Therapy in Primary and Metastatic Lung Adenocarcinoma (ID 3159)
17:29 - 17:40 | Author(s): M. Sadelain
- Abstract
Background:
Chimeric antigen receptor (CAR) T-cell therapy has shown durable remissions in hematological malignancies targeting cancer-antigen CD19. Ideal cancer-antigen targets for CAR T-cell therapy are antigens overexpressed on cancer cell-surface with limited expression in normal tissues, associated with tumor aggressiveness and expressed in a large cohort of patients. In our search for such candidate antigens in lung adenocarcinoma (ADC), we investigated the overexpression of Mesothelin (MSLN), MUC16 (CA125), and the combination of MSLN-MUC16 as the interaction of both antigens has been shown to play a role in tumor metastasis.
Methods:
In patients with stage I lung ADC (n = 912, 1995 - 2009), a tissue microarray consisting of 4 cores from each tumor and normal lung tissue was used to examine the antigen-expression characteristics, and their association with cumulative incidence of recurrence (CIR). Autologous metastatic tumor tissue was available from 36 patients. Differences in CIR between groups were tested using the Gray method (for univariate nonparametric analyses) and Fine and Gray model (for multivariate analyses).
Results:
MSLN and MUC16 were not expressed in normal lung tissue. In primary and metastatic lung ADC tumors, MSLN was expressed in 69% and 64%, MUC16 was expressed in 46% and 69%, both antigens were present in 50% and 33%, and either antigen were present in 33% and 49% respectively. On univariate analysis, patients with high MSLN expression had high risk of recurrence than low expression [5-year CIR, High: 25.1% vs Low: 17.6%, P = 0.017]. Patients with high MUC16 expression had high risk of recurrence than low expression [5-year CIR, High: 24.2% vs Low: 14.0%, P < 0.001]. Patients with high MUC16 and high MSLN had higher risk of recurrence than low expression [5-year CIR, High risk (High MUC16 and High MSLN): 27.6%, Intermediate risk (High MUC16 and Low MSLN): 24.2%, Low risk (Low MUC16): 13.6%, P < 0.001]. On multivariate analysis, increased MUC16-MSLN expression was associated with recurrence [Hazard ratio, 2.57 95% Confidence interval 1.41 – 4.68 P = 0.002], even after adjustment for currently known markers of lung ADC aggressiveness (gender, surgical procedure, stage, architectural grade and lymphatic invasion). High expression of MUC16 in the primary tumor was associated with high expression at recurrence sites.
Conclusion:
MSLN, MUC16 or a combination of expression of both antigens in patients with primary lung ADC is associated with increased risk of recurrence, a retained overexpression at metastatic sites in advanced lung ADC indicating that MUC16-MSLN expression is a marker of tumor aggressiveness. Expression in the majority of lung ADC patients imparting aggressiveness with no expression in normal lung provides the rationale to target MSLN and MUC16 for lung ADC CAR T-cell therapy.
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P3.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 226)
- Event: WCLC 2015
- Type: Poster
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.08-038 - Targetable Cancer-Associated Antigens for Immunotherapy in Malignant Pleural Mesothelioma (MPM) - Mesothelin, CA125 and WT-1 (ID 3165)
09:30 - 09:30 | Author(s): M. Sadelain
- Abstract
Background:
Mesothelin (MSLN), CA125 (also know as mucin-16, MUC16) and WT-1 are cancer-associated antigens currently under investigation as targets for tumor-specific immunotherapy, based on published observations that antigen-specific immune responses to these antigens prolong survival. In solid malignancies, we (Clin Cancer Res 2012, 2013) and others have published the role of MSLN in promoting tumor aggressiveness. Additionally, MSLN has been demonstrated to interact with CA125 in promoting invasion and metastasis, resulting in poor clinical outcomes. In this study, we investigated the individual and correlative expressions of MSLN, CA125 and WT-1 in both epithelioid and non-epithelioid MPMs.
Methods:
All available H&E-stained slides from patients who were diagnosed with MPM (1989-2010) were reviewed; tumors were classified according to the WHO classification. We constructed tissue microarrays (6 tumor cores/tumor) from 273 patients (epithelioid=224; non-epithelioid, including biphasic and sarcomatoid =49). MSLN, CA125, and WT-1 immunohistochemistry were performed, and total scores for each antigen were determined by assessing the combined intensity and distribution of the antigen expression.
Results:
Epithelioid MPMs demonstrated positive MSLN expression in 92% of patient samples (73% high-moderate expression), CA125 expression in 72% (19% high-moderate), and WT-1 in 94% (63% high-moderate) (Fig.1A). Triple positive antigen expression was recognized in 68% of patients; co-expression of two antigens was demonstrated in 23% of epithelioid MPMs (Fig.1B). In non-epithelioid MPMs, MSLN, CA125, and WT-1 were positive in 57% (16% high-moderate), 33% (2% high-moderate), and 98% (43% high-moderate) of patient tumors, respectively. Triple and double antigen co-expression were demonstrated in 29% and 33% of non-epithelioid MPMs, respectively. Only 1% of epithelioid and 2% of non-epithelioid MPMs demonstrated absence of expression of all three antigens: MSLN, CA125, and WT-1.Figure 1
Conclusion:
Our observation from a large cohort of MPM patients inclusive of all histological subtypes demonstrating greater than 98% positive expression of at least one of the three cancer-associated antigens in epithelioid and non-epithelioid MPMs, with strong antigen expression and high frequency of double and triple antigen expression, provides rationale to develop targeted therapies to these cancer-associated antigens for the treatment of MPM patients We are initiaiting a phase I clinical trial (NCT02414269) of mesothelin-targeted T-cell therapy for MPM patients at our center.
