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. 2014 Nov 5;6(261):261ra151.
doi: 10.1126/scitranslmed.3010162.

Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity

Prasad S Adusumilli  1 , Leonid Cherkassky  2 , Jonathan Villena-Vargas  2 , Christos Colovos  2 , Elliot Servais  2 , Jason Plotkin  3 , David R Jones  4 , Michel Sadelain  5
Affiliations

Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity

Prasad S Adusumilli et al. Sci Transl Med. .

Abstract

Translating the recent success of chimeric antigen receptor (CAR) T cell therapy for hematological malignancies to solid tumors will necessitate overcoming several obstacles, including inefficient T cell tumor infiltration and insufficient functional persistence. Taking advantage of an orthotopic model that faithfully mimics human pleural malignancy, we evaluated two routes of administration of mesothelin-targeted T cells using the M28z CAR. We found that intrapleurally administered CAR T cells vastly outperformed systemically infused T cells, requiring 30-fold fewer M28z T cells to induce long-term complete remissions. After intrapleural T cell administration, prompt in vivo antigen-induced T cell activation allowed robust CAR T cell expansion and effector differentiation, resulting in enhanced antitumor efficacy and functional T cell persistence for 200 days. Regional T cell administration also promoted efficient elimination of extrathoracic tumor sites. This therapeutic efficacy was dependent on early CD4(+) T cell activation associated with a higher intratumoral CD4/CD8 cell ratios and CD28-dependent CD4(+) T cell-mediated cytotoxicity. In contrast, intravenously delivered CAR T cells, even when accumulated at equivalent numbers in the pleural tumor, did not achieve comparable activation, tumor eradication, or persistence. The ability of intrapleurally administered T cells to circulate and persist supports the concept of delivering optimal CAR T cell therapy through "regional distribution centers." On the basis of these results, we are opening a phase 1 clinical trial to evaluate the safety of intrapleural administration of mesothelin-targeted CAR T cells in patients with primary or secondary pleural malignancies.

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Conflict of interest statement

Competing Interests: The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1. Regional administration of MSLN CAR-transduced T cells results in superior antitumor efficacy
(A) MSLN-targeted constructs with CD3ζ endodomain alone (Mz) or with the CD28 costimulatory domain (M28z). A PSMA-directed CAR with CD28 costimulation (P28z) as well as PSMA+-expressing EL4 targets are included in experiments as negative controls. Antigen-specific effector function of MSLN-CAR–transduced T cells as shown by lysis of MSLN-expressing, but not PSMA-expressing, target cells measured by chromium-release assays. (C and E) Tumor BLI of NOD/SCID/γcnull mice bearing pleural tumor. Tumor-bearing mice were treated with either 1×105 (1×) or 3×106 (30×) M28z T cells intravenously (E:T, 1:3000 or 100, respectively), compared with 1×105 (1×) or 3×105 (3×) M28z T cells intrapleurally (E:T, 1:3000 or 1000, respectively). Death is depicted by an asterisk (*). (D and F) Kaplan-Meier survival analysis demonstrates superior efficacy with intrapleural administration (solid blue line), compared with intravenous administration (dashed blue line). Median survival was not reached for intrapleural administration of M28z; median survival for intravenous administration was 27 days (1×) and 86 days (30×). Control mice treated with pleural P28z (black line) had a median survival of 27 to 42 days (n=4-10 per group). Survival curves were analyzed with Log-rank test. **P < 0.01; ***P < 0.001. Raw data and exact P values are provided in Supplementary Materials
Fig. 2
Fig. 2. Intrapleurally administered M28z+ T cells display early, robust proliferation of both CD4+ and CD8+ subsets
(A) Serial T-cell BLI in tumor-bearing mice. Intravenously administered M28z+ T cells display delayed but equivalent accumulation in the progressing pleural tumor. (B) Average effLuc-luciferase signal intensities from sequential T-cell BLI. Intrapleurally administered T cells (blue lines) display an earlier and sustained accumulation, with maximal T-cell signal at day 5. Intravenously administered T cells show delayed accumulation, with maximal signal at day 7. (C) E:T ratios reflect M28z T-cell accumulation in parallel with tumor burden at 6 h and days 1, 3, and 7, confirming the findings of T-cell BLI. Intravenous administration shows delayed T-cell accumulation, lower E:T ratios, and decreased CD8+ T cell infiltration. (D) FACS analysis at day 7 displays an equal accumulation of CD4+ and CD8+ T-cell subsets within the tumor and spleen after intrapleural administration, compared with decreased tumor accumulation of CD8+ T cells and equal distribution of CD4+ and CD8+ T cells in the spleen after intravenous administration. (E) A decrease in CD62L expression was observed in both CD4+ and CD8+ T cells following intrapleural administration. Error bars represent ±SEM. *P <0.05, **P < 0.01, ***P <0.001 by Student's t test. Raw data and P values are provided in the Supplementary Materials.
Fig. 3
Fig. 3. Intrapleurally administered M28z+ T cells display efficient systemic trafficking and accumulation in extrapleural tumor in an antigen-specific manner
(A) Serial tumor and T-cell BLI with dual luciferase imaging, demonstrating systemic trafficking and extrapleural tumor accumulation. Mice with established ffluc+ MSLN+ tumor in the right flank and pleural cavity and MSLN- tumor in the left flank received Gaussia-luciferase+ M28z T cells intrapleurally. A representative mouse with tumor in the flanks and pleural cavity before T-cell administration (left). T-cell BLI 15 days after T-cell administration (center) demonstrates residual T cells in the pleural cavity and accumulation in the MSLN+ right-flank tumor (center). One day later, tumor BLI shows a reduced burden in the MSLN+ right-flank tumor, compared with the MSLN- left-flank tumor (right). (B and C) Intrapleurally administered M28z+ T cells show early and robust accumulation in MSLN+ intraperitoneal tumor, compared with intravenously administered T cells. (C) Quantification of the fold increase in signal intensity of the peritoneal cavity in tumor-bearing mice displays enhanced T-cell accumulation with intrapleural administration, compared with intravenous administration (n=3 per group, error bars represent ±SEM. Raw data and P values are provided in the Supplementary Materials).
Fig. 4
Fig. 4. Intrapleurally administered M28z T cells eradicate pleural tumor and establish long-term CD4+ predominant persistence
(A) CD28 costimulation facilitates tumor eradication following a single dose of T cells. In total, 1×105 CAR+ Mz, M28z, or P28z (negative control) T cells were intrapleurally administered into mice bearing established tumors. (Left) Tumor burden. (Right) Kaplan-Meier survival curve. Median survival of the Mz and M28z groups (at least 9 mice per group) was 63 days and median survival not reached, respectively. Survival curve was analyzed by log-rank test. **P<0.01. (B) CD28 costimulation enhances CAR+ T-cell persistence. Absolute CAR+ T-cell counts (per mL of peripheral blood) at 50 days after intrapleural administration of 3×106 CAR+ T cells. Error bars represent ±SEM, *P < 0.05 by Student's t test. (C) Persisting CAR+ T cells are predominantly CD4+. (Left) FACS analysis of peripheral blood in treated mice. (Right) CD4:CD8 ratios determined at successive time points following T-cell administration. The preinfusion CD4:CD8 ratio was approximately 0.5. Results shown are similar across a range of T-cell doses (3×106, 1×106, and 3×105 CAR+, n=3 mice at each time point). *P<0.05 by Student's test with Bonferroni correction for multiple comparisons comparing ratio at each time point to the preinfusion CD4:CD8 ratio. Raw data and P values are provided in the Supplementary Materials.
Fig. 5
Fig. 5. Functional persistence of intrapleurally administered MSLN CAR T cells is predominantly mediated by CD4+ and is augmented by CD28 costimulation
(A) FACS analysis of splenic single-cell suspension prepared from mice (n=3) sacrificed 202 days after tumor eradication, following a single dose of 3×105 M28z T cells administered intrapleurally. CD3+GFP+M28z T cells show a predominance of effector memory CD4+ T cells. (B) Tumor BLI of mice rechallenged with MSLN+ and MSLN- tumor. Eighty-seven days after pleural tumor eradication, following administration of a single dose of 3×105 M28z or Mz T cells, 1×106 MSLN+ or MSLN- tumor cells were injected into the peritoneal cavity. Following tumor rechallenge, Mz T cells prevent tumor growth, whereas M28z T cells promote tumor regression. (C and D) Absolute M28z or Mz T-cell numbers in the spleen after tumor rechallenge with either MSLN+ (n=6) or MSLN- (n=6) tumor; mice were sacrificed 16 days after tumor rechallenge. Only the M28z T-cell–treated mice rechallenged with MSLN+ tumor show a robust accumulation of CAR+ T cells in the spleen, the majority of which are CD4+ T cells. Error bars represent ±SEM, raw data and P values are provided in the Supplementary Materials.
Fig. 6
Fig. 6. CD4+ M28z T cells augment CD8+ accumulation that is enhanced with preactivation
(A–C) Unsorted M28z and Mz or bead-sorted CD4+ and CD8+ T cells were assayed. M28z CD4+ T cells show (A) higher cytokine secretion (from 4- to 14-fold; ***P<0.001 by Student's t test) and (B) profound T-cell expansion without exogenous IL-2. (C) CD4+ M28z activation facilitates robust CD8+ M28z T-cell accumulation upon repeated antigen stimulation in vitro. (D) Antigen-activated CD4+ M28z activation facilitates robust CD8+ M28z T-cell accumulation in vivo. Isolated CD8+ effLuc M28z T cells were intrapleurally administered to MSLN+ pleural tumor–bearing mice with either CD4+ M28z (n=6) or CD4+ control–transduced T cells (n=6) and were serially imaged. One representative mouse (n=6 per group; left) displays increased CD8+ M28z T-cell accumulation in the presence of CD4+ M28z. (E) The average accumulation of CD8+ CAR+ T cells was calculated at the indicated intervals (P values as shown calculating fold increase from 16 to 72 hours, n=6 per group). (F) Preactivation of M28z CD4+ enhances CD8+ proliferation, compared with simultaneous activation of CD4+. Bead-sorted CD8+ Mz or M28z T cells were cocultured with either corresponding Mz or M28z CD4+ or preactivated CD4+ T cells (activated on MSLN+ tumor cells 24 h before the assay). Preactivation of M28z CD4+ enhances the accumulation of CD8+ to a greater degree than does CD8+ and CD4+ concurrent stimulation.
Fig. 7
Fig. 7. CD4+ MSLN CAR+ T cells demonstrate efficient cytolytic function that is granzyme/perforin dependent
(A) CD4+ M28z T cells show a delayed but similar cytotoxicity as CD8+ M28z T cells. (B) CD28 costimulation enhances CD4+-mediated cytotoxicity. (C) Cytokine-rich supernatants obtained from stimulated CD4+ M28z CAR+ T cells enhance cytotoxicity of both CD8M28z and CD4M28z T cells. (D) CAR T-cell lytic function is dependent on release of cytotoxic granules. Bulk, CD4, or CD8 M28z and Mz T cells were cocultured for 18 h in the presence or absence of the chelating agent ethylene glycol tetraacetic acid (EGTA). (A–D) Cytotoxicity of bead-purified CD4+ or CD8+ Mz and M28z T cells. (E, left) CD4+ CAR T cells express granzyme B, but with delayed kinetics, compared with CD8+ CAR T cells. Intracellular FACS analysis for granzymes B was performed on resting PBMCs, PHA-stimulated blasts, and M28z, Mz, and P28z T cells stimulated with MSLN+ for 4 or 18 h.(E, right) CD28 costimulation enhances granzyme B expression. Histograms show expression at 18 h after MSLN+ stimulation. Error bars represent ±SEM, *P < 0.05 by Student's t test. Raw data and P values are provided in the Supplementary Materials.
Fig. 8
Fig. 8. Intrapleurally administered CD4+ M28z CAR T cells are efficacious when administered alone in vivo; mediate enhanced efficacy, compared with CD8+ M28z T cells; and establish long-term functional persistence
(A) BLI tracking the progression of tumor burden. Eighteen days after tumor injection, mice received either 3×105 (3x), 1×105 (1x), or 3×104 (0.3x) CAR+ T cells of bulk M28z (n=5), bead-sorted CD4+, CD8+ M28z (n=7), or P28z (n=4). (B) Kaplan-Meier survival curves. At all doses, CD4+ M28z CAR+ T cells were efficacious, compared with CD8+ CAR+ T cells. The antitumor efficacy of CD4+ CAR+ T cells was comparable to that of unsorted CAR+ T cells. *P < 0.05; **P < 0.01; ***P < 0.001 by Student's t test. Raw data and P values are provided in the Supplementary Materials. (C) Tumor BLI of mice rechallenged with tumor. At 196 days after intrapleural administration of a single dose of 3×105 (3x) unsorted (bulk) M28z or CD4+-sorted M28z T cells, 1×106 MSLN+ tumor cells were injected into the peritoneal cavity. Persisting CD4+ M28z T cells prevented tumor growth.
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