Personalized neoantigen pulsed dendritic cell vaccine for advanced lung cancer

Lung cancer is one of the malignant tumors with the fastest growth in morbidity and mortality and poses a major threat to human health. Surgery and definitive chemoradiotherapy remain standard for early-stage disease, while targeted therapies and immune checkpoint inhibitors (ICIs) have improved outcomes for some patients with advanced disease. However, patients who fail frontline therapies have limited options and poor prognosis, underscoring the need for novel, effective, low-toxicity treatments. Over recent decades, multiple immunotherapy strategies have emerged, including ICIs, adoptive cell therapy (ACT), and cancer vaccines. A growing body of evidence indicates that tumor-specific neoantigens, arising from somatic mutations, underlie responses to ICIs and ACT, and TILs targeting neoantigens can mediate tumor regression. Neoantigen-based RNA or peptide vaccines have shown promising effects in melanoma and glioblastoma. Dendritic cells (DCs) are key antigen-delivery vectors, and antigen-loaded DC vaccines can induce stronger immune responses than antigen-plus-adjuvant formulations. Neoantigen-based DC vaccines have shown clinical success in melanoma and other solid tumors. Given lung cancer’s high tumor mutational burden and neoantigen load, neoantigen-based DC vaccination is a rational option, although no prospective lung cancer–focused trials had been published. This study conducted a pilot, single-arm, multicenter trial to investigate the safety and efficacy of a personalized neoantigen peptide–pulsed autologous DC vaccine (Neo-DCVac) in advanced lung cancer. Preliminary results indicated Neo-DCVac is feasible, safe, and effective, capable of eliciting antigen-specific T-cell responses and antitumor immunity.

Prior work shows that neoantigens drive effective antitumor immunity and contribute to clinical benefit from ICIs and ACT. Adoptive transfer of neoantigen-specific TILs has achieved meaningful tumor regression across cancers. Personalized neoantigen vaccines, including RNA and peptide platforms, have induced robust immune responses and clinical signals in melanoma and glioblastoma. DC-based vaccines, as professional antigen-presenting cell platforms, can outperform peptide-plus-adjuvant vaccines in immunogenicity and have demonstrated clinical activity in melanoma and refractory solid tumors. Preclinical and early clinical data support combining DC vaccines with checkpoint blockade (e.g., CTLA-4 inhibitors) to enhance T-cell antitumor activity. Lung cancer’s high mutational burden suggests a rich neoantigen landscape, supporting exploration of neoantigen-targeted DC vaccination in this disease.

Study design and oversight: Prospective, single-arm, pilot study conducted at two medical centers in China (West China Hospital and Shanghai Tenth People’s Hospital, Tongji University). Ethics approval obtained (2016-27). Registered at ChiCTR-ONC-16009100 and NCT02956551. Written informed consent required. Patients: Adults (18–75) with histologically/cytologically confirmed lung cancers (squamous cell carcinoma, adenocarcinoma, neuroendocrine tumor, or SCLC), at least one measurable lesion per RECIST v1.1, ECOG 0–1, adequate organ function, life expectancy ≥3 months. Progression after standard therapies required; washout: >4 weeks for chemotherapy and >2 weeks for targeted therapy; prior ICI exposure allowed without washout. Key exclusions: second primary cancer, unstable comorbidities (e.g., active ulcer, grade 3 hypertension, unstable angina, CHF, active hepatitis, uncontrolled infection), >10% weight loss in 6 weeks, chronic corticosteroids, and tumors too difficult to biopsy. Sample collection and sequencing: Fresh tumor biopsies (various sites) and EDTA-anticoagulated blood were collected. DNA/RNA extracted (Qiagen kits) and quality-assessed. Trio samples (tumor DNA, tumor RNA, matched normal DNA) underwent WES (Agilent SureSelect Human All Exon 44 Mb v2.0) and RNA-seq (Illumina TruSeq RNA Access). Sequencing on Illumina HiSeq X Ten, 150 bp paired-end. DNA reads aligned with BWA-MEM to GRCh37/hg19; duplicates removed, indel realignment, base recalibration via GATK. RNA reads aligned with STAR; duplicates removed and splice junction handling via GATK. Somatic variant calling and expression: Somatic mutations called using Mutect2 (tumor-normal pairs) with minimum coverage and VAF thresholds (≥10 reads in tumor/normal, ≥10% tumor VAF, distinct pairs). Variants annotated (SnpEff/SnpSift), filtered to rare (<0.001 in gnomAD East Asian), coding, nonsynonymous SNVs or in-frame indels. Manual IGV review applied. Copy-number (CNVkit), tumor purity/ploidy (ABSOLUTE), and clonal structure (PyClone) analyzed; clonal if CCF >0.8. Gene expression quantified with featureCounts (Gencode v19). RNA mutant allele frequencies called with GATK HaplotypeCaller. HLA typing and LOH: HLA alleles inferred from tumor exome reads using Polysolver and HLAminer; HLA loss of heterozygosity assessed by comparing tumor with germline typing. Neoantigen prediction and selection: For class I, all variant-containing 8–14-mers; for class II, 15-mers. Binding affinity predicted using IEDB tools (recommended mode). Candidate neoepitopes required predicted HLA binding affinity <500 nM. Selection prioritized: (i) strong binders (IC50 <50 nM or %Rank <0.5); (ii) mutant stronger than WT; (iii) higher tumor VAF; (iv) RNA support (>5 mutant reads); (v) oncogenic mutations prioritized. Practical peptide properties (e.g., solubility, cysteines) considered. Per patient, 13–30 peptides chosen for synthesis. Peptide synthesis: 5–15 aa peptides synthesized by solid-phase methods; purified by RP-HPLC (Shimadzu LC-20AT; solvents: 0.1% TFA in water/acetonitrile; 1 ml/min; 214 nm; SHIMADZU Inertsil ODS-SP 4.6×250 mm, 5 μm). DC generation and vaccine manufacture: PBMCs collected by leukapheresis (COM.TEC) and processed in GMP lab. Monocyte-derived DCs generated via adherence in AIM-V with 1% autologous serum, GM-CSF (1000 IU/ml) and IL-4 (500 IU/ml). On day 5, immature DCs pulsed with neoantigen peptides (50 μg/ml) for 20–24 h, then matured 20–24 h with TNF-α (10 ng/ml), IL-1β (10 ng/ml), IFN-γ (1000 U/ml), PGE2 (250 ng/ml), R848 (1 μg/ml), and poly(I:C) (20 ng/ml). Quality testing included flow cytometry for HLA-DR/ABC, CD11c, CD1c, CD141, CD54, CD70, CD80, CD83, CD86, CCR7, and PD-L1; IL-12p70 secretion by CBA; sterility (mycoplasma, bacteria/fungi) and endotoxin (<5 EU/ml). Mature DCs harvested, washed, counted; 1–3×10^6 DCs in 2.5 ml normal saline with 1% human albumin per clinical dose. First dose administered fresh; remaining doses (5–10×10^5 DCs per dose) cryopreserved and thawed 2–3 h before administration. Treatment regimen: Preconditioning cyclophosphamide 250 mg/m^2 on day −1. Subcutaneous Neo-DCVac administered bilaterally to axillary and inguinal regions on day 0. GM-CSF 0.075 mg subcutaneously daily on days 1–5. Vaccination cycle: repeat Neo-DCVac at weeks 1, 2, 4, 6, and 8; if no progression, subsequent cycles permitted; upon progression, switch to other therapies or best supportive care. Outcome measures: Safety assessed by CTCAE v4.0. Tumor response by RECIST v1.1 at end of each vaccination cycle (CT chest/upper abdomen, brain MRI, bone scan). PFS from therapy start to progression, intolerable toxicity, or death; OS from therapy start to death. Immune monitoring: Autologous PBMCs collected after each cycle. In vitro stimulation with mutant/WT peptides (10 μg/ml) or peptide-pulsed DCs (DC:PBMC 1:10) in AIM-V + 10% FBS with IL-15 (5 ng/ml); IL-2 (10 U/ml) added day 3; restimulation after 10–21 days. Assays included IFN-γ ELISpot (positive if >2× negative control or WT), activation markers (CD134/OX40, CD137/4-1BB) by flow cytometry, intracellular cytokine staining (IFN-γ, IL-2, TNF-α, CD107a) after overnight peptide exposure, and cytokine secretion by CBA. For selected patients, TCRβ CDR3 sequencing performed after peptide incubation to assess diversity, clone frequency, and convergence. Statistics: Descriptive statistics for baseline and safety; Kaplan–Meier for PFS/OS with 95% CIs; two-tailed t-tests for group comparisons; significance at p<0.05. GraphPad Prism 8 used.

- Enrollment and feasibility: 18 advanced lung cancer patients recruited (Nov 2017–Sep 2019) across two centers; 17 underwent sequencing; 5 excluded (insufficient neoepitopes n=2; HLA LOH n=1; rapid progression/death n=2); 12 treated. Median time from biopsy to first vaccination 2.8 months (range 2.1–3.5). Manufacturing feasible for all; 17 leukapheresis events yielded median 2.55×10^6 PBMCs (range 4.14×10^6–1.50×10^7) and median 1.80×10^8 DCs (range 0.70×10^8–4.33×10^8); median DC yield from PBMCs 8.37% (range 2.73–20.83%). Total 85 Neo-DCVac doses produced; median 5 per patient (range 3–14). Median viable cell percentage 81% (71–94%); viable cells per dose median 1.60×10^7 (0.65×10^7–2.4×10^7). Mature DC phenotype: CD11c/CD86 >90%, CD83 >70%, CD11b/CD209 >90%, CCR7 >40%. Mature DCs secreted significantly more IL-12p70 than immature DCs; sterility and endotoxin acceptable (<5 EU/ml). - Neoantigen selection: Median 312 (range 80–808) somatic nonsynonymous mutations per patient. For each patient, 13–30 peptides selected (12–30 noted elsewhere); overall mutational landscape showed frequent TP53 alterations; EGFR mutations in 1 patient. - Safety: All treatment-related adverse events grade 1–2; no dose-limiting toxicities, dose delays, or discontinuations due to toxicity. Injection-site reactions in 12/12 (100%). Grade 1 neutropenia in 1/12 (8.3%); grade 2 rash in 1/12 (8.3%). No increase in ICI-related immune adverse events in patients receiving concurrent ICIs. - Efficacy: Median follow-up 7.1 months (0.9–17.2). Median PFS 5.5 months (95% CI 1.9–9.2). Median OS 7.9 months (95% CI 5.9–10.0). Objective response rate 25% (3/12 PR; 0 CR). Disease control rate 75% (9/12). Six of 12 patients had tumor target lesion reduction; three patients (25%) had PD. Notable cases included PR in a heavily pretreated adenocarcinoma (patient 1, PR 7.6 months), durable control of brain metastases in EGFR L858R adenocarcinoma (>1 year, patient 7), and prolonged SD in extensive-stage SCLC (~11.2 months, patient 15). - Combination with ICIs: Four patients who had primary non-response or relapse on ICIs achieved disease control after adding Neo-DCVac (2 PR, 2 SD), with up to 80% tumor reduction. Prespecified comparison suggested longer PFS with combined Neo-DCVac+ICIs vs Neo-DCVac alone (median 11.2 vs 2.2 months; p=0.045) and a trend toward longer OS (median 11.2 vs 7.6 months; p=0.40); small sample size limits inference. - Immunogenicity: In 10 evaluable patients with post-vaccination PBMCs, antigen-specific T-cell responses to predicted neoantigens were consistently observed by ELISpot/flow/CBA. In patient 1, post-vaccination PBMCs showed increased IL-2, IFN-γ, TNF-α secretion and mutant-specific CD4+/CD8+ responses; responding CD4+ T cells expressed CD45RO and PD-1 (memory phenotype). In patient 15 (SCLC), stronger mutant vs WT responses in PBMCs and increased CD134+ CD4+ T cells; cytokines increased; TCR repertoire showed decreased diversity, increased mean clone frequency and convergence after mutant peptide stimulation, with >1000-fold expansion of specific clones. In patient 17, tumor burden reduced by 29% with mutant-specific PBMC responses. - Correlative insight: Among six patients with >8 neoantigens eliciting T-cell responses, 1 had an objective response and 5 had disease control; among four patients with <8 responsive neoantigens, 50% progressed, suggesting breadth of immunogenic neoantigens may relate to clinical outcome (limited by small numbers).

The study demonstrates that a personalized neoantigen peptide–pulsed autologous dendritic cell vaccine (Neo-DCVac) is feasible, safe, and capable of inducing antigen-specific T-cell responses in heavily pretreated advanced lung cancer patients. Clinical activity was observed with an ORR of 25% and DCR of 75% despite poor baseline prognostic features, and with manageable, low-grade toxicities. Importantly, combining Neo-DCVac with ICIs appeared to enhance clinical benefit, consistent with mechanistic synergy wherein checkpoint blockade augments T-cell activation primed by mature DCs. The observation that Neo-DCVac could reinduce responses in patients relapsing on anti–PD-1 therapy suggests potential for overcoming or modulating resistance. Immunomonitoring confirmed vaccine-induced, neoantigen-specific CD4+ and CD8+ T-cell responses, increased cytokine production, memory phenotypes, and expansion of specific TCR clones, supporting on-target immune activation. A preliminary relationship between the number of immunogenic neoantigens and disease control was noted, indicating that broader T-cell response breadth may translate into improved outcomes. These findings address the unmet need for effective, low-toxicity options in advanced lung cancer and provide first prospective clinical evidence supporting neoantigen-based DC vaccination in NSCLC. They also reinforce a biologically plausible strategy of combining personalized vaccination with checkpoint inhibitors to maximize antitumor immunity.

Personalized neoantigen peptide–pulsed DC vaccination (Neo-DCVac) is a feasible and safe approach for heavily pretreated advanced lung cancer, eliciting robust neoantigen-specific T-cell responses and showing preliminary clinical efficacy (ORR 25%, DCR 75%, median PFS 5.5 months, median OS 7.9 months). Combination with ICIs demonstrated signs of synergistic benefit, including in patients with prior ICI resistance. These results provide initial prospective evidence supporting neoantigen-based DC vaccines in NSCLC and potentially other solid tumors. Future directions include larger controlled trials to validate efficacy and define the benefit of combination strategies; optimization of neoantigen prediction and selection (e.g., improved bioinformatics, integration of proteomic presentation data); exploration of whole-tumor lysate–loaded DCs; and deeper correlative studies to link immune response breadth and phenotype (CD4+/CD8+) with clinical outcomes.

- Small sample size (n=12 treated) and single-arm, non-randomized design limit efficacy inference and generalizability. - Heterogeneous histologies (NSCLC subtypes and SCLC), prior therapies (including ICIs), and concurrent treatments introduce confounding. - Median 2.8-month interval from biopsy to first vaccination may impact patient selection and outcome due to disease dynamics. - In silico neoantigen prediction accuracy is limited; no clear correlation between predicted HLA affinity and observed T-cell responses was established. - Limited immune correlative analyses across the cohort (detailed CD4+/CD8+ response characterization in only a subset) constrain conclusions about response correlates. - Lack of complete responses and short follow-up for some patients preclude assessment of long-term durability. - Manufacturing and logistics considerations (multi-peptide synthesis, GMP DC production) may affect scalability.