Phase I Trial of Intratumoral Injection of CCL21 Gene-Modified Dendritic Cells in Lung Cancer Elicits Tumor-Specific Immune Responses and CD8+ T-cell Infiltration

Abstract

Purpose: A phase I study was conducted to determine safety, clinical efficacy, and antitumor immune responses in patients with advanced non-small cell lung carcinoma (NSCLC) following intratumoral administration of autologous dendritic cells (DC) transduced with an adenoviral (Ad) vector expressing the CCL21 gene (Ad-CCL21-DC). We evaluated safety and tumor antigen-specific immune responses following in situ vaccination (ClinicalTrials.gov: NCT01574222).Experimental Design: Sixteen stage IIIB/IV NSCLC subjects received two vaccinations (1 × 106, 5 × 106, 1 × 107, or 3 × 107 DCs/injection) by CT- or bronchoscopic-guided intratumoral injections (days 0 and 7). Immune responses were assessed by tumor antigen-specific peripheral blood lymphocyte induction of IFNγ in ELISPOT assays. Tumor biopsies were evaluated for CD8+ T cells by IHC and for PD-L1 expression by IHC and real-time PCR (RT-PCR).Results: Twenty-five percent (4/16) of patients had stable disease at day 56. Median survival was 3.9 months. ELISPOT assays revealed 6 of 16 patients had systemic responses against tumor-associated antigens (TAA). Tumor CD8+ T-cell infiltration was induced in 54% of subjects (7/13; 3.4-fold average increase in the number of CD8+ T cells per mm2). Patients with increased CD8+ T cells following vaccination showed significantly increased PD-L1 mRNA expression.Conclusions: Intratumoral vaccination with Ad-CCL21-DC resulted in (i) induction of systemic tumor antigen-specific immune responses; (ii) enhanced tumor CD8+ T-cell infiltration; and (iii) increased tumor PD-L1 expression. Future studies will evaluate the role of combination therapies with PD-1/PD-L1 checkpoint inhibition combined with DC-CCL21 in situ vaccination. Clin Cancer Res; 23(16); 4556-68. ©2017 AACR.

Conflict of interest statement

Conflict of interest disclosure: None

©2017 American Association for Cancer Research.

Figures

Figure 1. Protocol summary and generation of adenoviral CCL-21 transduced DC from lung cancer patients

(A) Patients were assessed for eligibility by checking baseline tumor burden and underwent leukapheresis if eligible. PBMC were isolated from the leuko pak and cryopreserved until use and an aliquot was used for HLA typing. Patients received intratumoral injection of vaccine twice with a one-week interval in between. Needle biopsy was performed just prior to injection on the same day (Protocol day 0 and day 7) to allow collection of samples from the tumor. Peripheral blood samples were drawn for safety monitoring (at Screening and on days 0, 7, 14, and 28) and immune monitoring studies (days 0, 7, 28, and 56). (B) DC were generated following 6 days of culture with GM-CSF and IL-4 from cryopreserved mononuclear cells (MNC) obtained by leukapheresis, as described in Materials and Methods. The summary of the yield of MNC and DC, recovery of DC before adenoviral transduction (BT) and after adenoviral transduction (AT) with AdCCL21 and viability by trypan blue staining is reported. (C) After a 6 day culture of MNC in the presence of GM-CSF and IL-4, cells were analyzed for surface markers such as CD86, HLD-DR, CCR7, CD83, CD80, CD14, CD19 and CD3 by flow cytometry, as described in Materials and Methods. The results were expressed as a representative phenotype in 1 of 16 patients. (D) and (E) Summaries of the phenotypic results from 16 patients are shown. Note that there are two data points from 2 preparations of the DC culture per one patient. LK; leuko pak, BC; before cryopreservation, AC; after cryopreservation, BT; before transduction, AT; after transduction, *; cell number used for DC culture,**; (DC yield/MNC Thawed) x100, LGC; large granular cells

Figure 2. Tumor associated antigen (TAA) expression profiles in NSCLC patients and HLA restricted synthetic peptides of TAA used for IFN-γ ELISPOT assay

(A) Lung tumor biopsy for 16 patients was performed on day 0 and day 7 after the vaccine administration and tumor associated antigen expression profiles were determined by qRT-PCR using the tumor antigen panel as described in Material and Methods. Over expression of TAA was defined as an expression of more than 100 gene copies per 106 β-actin gene copies. Percentage of patients that overexpress each TAA were shown. (B) Patients’ HLA matched tumor associated antigen derived synthetic peptides were selected and added to the 96 well plate of INF-γ ELISPOT assay. Six of 16 patients showed immune responses to TAA specific INF-γ production.

Figure 3. Immunologic responses to vaccination

PBMC were collected pre- and post-vaccination and were co-cultured with patient’s HLA matched peptides and derived from tumor associated antigens for 24 hours to monitor immune responses by IFN-γ ELISPOT assay as described in Materials and Methods. (A and B) Six of 16 patients showed vaccine dependent response to IFN-γ production. (B) Three of 6 responders (SLC01, SLC07, and SLC28) had a high response with TAA non-specific and vaccine independent IFN-γ production at baseline that declined after vaccination yet met criteria for TAA-specific and vaccine dependent immune responses. Profiles of tumor associated antigen for each patient are shown in the lower panel.

Figure 4. Association between PD-L1 expression and IFN-γ ELISPOT assay response or CD8+ T cells infiltration into tumor

(A) Left; PD-L1 gene copy numbers were compared between patients with (n=4) and without (n=9) IFN-γ induction on days 0 and 7 after vaccine administration. Right; PD-L1 gene copy numbers were compared between patients with (n=4) and without (n=9) tumoral infiltration of CD8+ T cells on day 0 and day 7 after vaccine administration. (B) A summary of IFN-γ ELISPOT assay response on day 14, 28 or 56 after vaccine administration, and tumoral infiltration of CD8+ T cells on day 0 and day 7 after vaccine administration. Results are shown from a total of 16 patients are shown.

Figure 5. Tumor immunohistochemical staining with CD8 and PD-L1

(A) Paraffin-embedded tumor tissues were stained with anti-CD8 and anti-PD-L1 on day 0 and day 7 and representative immunohistochemical staining images from SLC12 are shown. (B) Representative immunohistochemical staining images of membranous and cytoplasmic expression of PD-L1 on day 0 from patients SLC23 and SLC 09, respectively, are shown.