Peripheral Blood ETASTs for Predicting Efficacy of Chemoimmunotherapy in NSCLC

Prospective Study of Changes in Peripheral Blood Effector Tumor Antigen-Specific T Cells for Predicting Efficacy of Chemoimmunotherapy in Non-Small Cell Lung Cancer

The goal of this observational study is to explore whether changes in peripheral blood effector tumor antigen-specific T cells (ETASTs) can predict treatment outcomes in patients with advanced non-small cell lung cancer (NSCLC) receiving chemoimmunotherapy. The study aims to:

• Evaluate the relationship between ΔETAST levels (baseline to cycle 2) and progression-free survival
• Compare the predictive performance of ΔETASTs with traditional biomarkers (PD-L1, TMB)
• Assess whether ΔETASTs can identify patients more likely to benefit from PD-1 inhibitor plus chemotherapy

Participants will:

• Provide peripheral blood samples at baseline and after cycle 2 of treatment
• Undergo ETAST quantification using the CTT-NanoDT technology with TATAN nanoparticles
• Have standard tumor assessments every 2 cycles according to RECIST 1.1 criteria
• Be followed for progression-free survival and overall survival up to 24 months

Non-small cell lung cancer (NSCLC) accounts for the majority of lung cancer cases globally. While immune checkpoint inhibitors (ICIs), particularly PD-1/PD-L1 inhibitors combined with platinum-based chemotherapy, have become standard first-line therapy for advanced NSCLC, only a subset of patients achieve clinical benefit. Current biomarkers including PD-L1 expression and tumor mutational burden (TMB) have limited predictive accuracy, creating an urgent need for more reliable biomarkers.

Tumor antigen-specific T cells (TASTs) are the actual effectors in ICI therapy, and successful ICI response depends on reactivation of these cells. However, detection of circulating tumor antigen-specific T cells (CTASTs) has been technically challenging due to their low frequency and heterogeneity in peripheral blood.

This study utilizes a novel Circulating Tumor-Specific T Cell Nanodetection Technology (CTT-NanoDT) developed by the research team. The technology employs Tumor Antigen-specific T cell Activating Nanoparticles (TATAN) loaded with whole tumor cell components to specifically activate and quantify effector TASTs (ETASTs) in peripheral blood.

Study Design:

This is a prospective, single-center, observational cohort study enrolling 80 patients with stage IIIB-IV NSCLC receiving standard PD-1 inhibitor plus platinum-based chemotherapy. Peripheral blood samples (5 mL) will be collected at two time points: baseline (T0, within 1 day before treatment initiation) and after completion of cycle 2 (T1, day 21 of cycle 2).

ETAST Detection Protocol:

1. PBMC isolation from peripheral blood using Ficoll density gradient centrifugation
2. Co-incubation of PBMCs with TATAN nanoparticles (50 μg/mL) for 48 hours at 37°C
3. Flow cytometric quantification of activated ETASTs defined as CD3+CD8+IFN-γ+ and CD3+CD8+CD137+ double-positive cells
4. Calculation of ΔETAST: [(ETASTs-T1 - ETASTs-T0) / ETASTs-T0] × 100%

Primary Endpoint:

Progression-free survival (PFS), defined as time from treatment initiation to first documented disease progression per RECIST 1.1 or death from any cause, assessed by independent radiology committee.

Secondary Endpoints:

1. Comparison of predictive performance (ROC curve AUC) between ΔETASTs and traditional biomarkers (PD-L1 TPS, TMB) using DeLong test
2. Objective response rate (ORR), overall survival (OS), disease control rate (DCR)
3. Treatment-related adverse events graded per CTCAE 5.0

Statistical Analysis:

Cox proportional hazards regression will assess the association between ΔETASTs and PFS. Patients will be stratified by median ΔETAST value into high and low change groups for survival comparisons using Kaplan-Meier curves and log-rank tests. Sample size of 80 provides 80% power to detect HR=0.42 with α=0.05, accounting for 15% dropout rate.

The study aims to establish ΔETASTs as a superior biomarker for predicting chemoimmunotherapy efficacy in NSCLC, potentially enabling precision patient selection and improving treatment outcomes.

Inclusion Criteria:

• Histologically or cytologically confirmed stage IIIB-IV non-small cell lung cancer (NSCLC)
• Planned to receive PD-1 inhibitor combined with platinum-based chemotherapy (e.g., pembrolizumab + pemetrexed/carboplatin)
• Age 18-80 years
• ECOG performance status 0-1
• Expected survival ≥12 weeks
• Adequate bone marrow function: ANC ≥1.5×10⁹/L, PLT ≥100×10⁹/L
• Adequate hepatorenal function: Cr ≤1.5×ULN, ALT/AST ≤2.5×ULN
• At least one measurable lesion per RECIST 1.1 criteria
• Able to provide informed consent and comply with study procedures including serial blood sampling and imaging follow-up

Exclusion Criteria:

• No measurable disease per RECIST 1.1 criteria
• Tumor emergencies requiring immediate intervention (spinal cord compression, superior vena cava syndrome)
• Active untreated central nervous system metastases or leptomeningeal disease
• Prior treatment with immune checkpoint inhibitors within 4 weeks before enrollment
• Chronic use of immunosuppressive agents (e.g., corticosteroids >10 mg/day prednisone equivalent)
• Coagulation disorders (INR >1.5 or APTT >1.5×ULN) or ongoing anticoagulation therapy
• Poor vascular access precluding serial venipuncture (>5 mL per draw)
• Active hepatitis B (HBV DNA >2000 IU/mL), hepatitis C, or HIV infection
• Uncontrolled bacterial or fungal infection requiring systemic treatment
• Pregnancy or lactation
• Severe psychiatric disorder or communication barriers affecting informed consent or follow-up compliance

Withdrawal Criteria:

• Participant voluntary withdrawal with signed withdrawal statement
• Major protocol violations: failure to receive ≥2 cycles of planned chemoimmunotherapy; missing ≥2 critical timepoint blood samples (baseline, cycle 2)
• Uncontrollable grade ≥3 immune-related adverse events requiring permanent discontinuation of PD-1 inhibitor

Study Termination Criteria:

• Disease progression confirmed by imaging per RECIST 1.1 or clinical progression requiring radiotherapy
• Death or loss to follow-up >6 months
• Unacceptable grade 4 treatment-related toxicity
• Investigator determination that continued participation poses health risk to patient
• Study terminated by ethics committee for scientific or administrative reasons