Title : Patient-derived cell lines from Vietnamese leukemia patients: Whole-genome landscape, mechanism, and HLA-personalized multi-epitope neoantigen vaccine design

Background and objectives: Leukemia cell lines of Southeast Asian origin remain extremely rare in international biorepositories, and personalized neoantigen vaccines for hematologic malignancies are still barely implemented in middle-income countries. This study aimed to (i) generate and characterize patient derived cell lines from Vietnamese leukemia patients, (ii) explore the genetic landscape and driver modules of a representative line, (iii) evaluate response to standard chemotherapy, and (iv) design and computationally evaluate HLA-personalized multi-epitope vaccines.

Methods: Thirty patients were enrolled per WHO 2022 and ICC 2022 criteria; bone marrow blasts were immortalized in vitro. The representative line AL035 (CMML) underwent whole-genome sequencing (WGS, ≥30×) and interaction-network analysis with STRING/Reactome. Drug sensitivity was assessed by IC₅₀ for cytarabine (Ara-C) and daunorubicin (DNR), with combination effects analyzed in SynergyFinder (Bliss and Loewe models). Epitopes predicted with NetMHCpan-4.1/NetMHCIIpan-4.3 were assembled into two constructs, modeled with AlphaFold2/I-TASSER, tested for stability by molecular dynamics in GROMACS (100 ns), and for immunogenicity by C-ImmSim v10.1 (350 days).

Results: Five stably immortalized cell lines were established (success rate 16.7%; 95% CI 7.4–33.5%) - the first Vietnamese leukemia cell-line panel reported internationally. WGS of AL035 identified 13,298 structural variants and 681 CNVs; network analysis defined a 9-gene module converging on FOXP1, suggesting a three-axis model of epigenetic dysregulation–mutation accumulation–immune evasion. AL035 showed moderate sensitivity to Ara-C (IC₅₀ = 0.055 µM) and DNR (IC₅₀ = 0.024 µM), comparable to reference line U937 and consistent with the drug-resistant CMML phenotype. The Ara-C + DNR combination was strongly synergistic at low concentrations (ΔBliss = 43.9%; Loewe CI = 0.01), indicating dose-sparing potential for elderly patients and a rationale for combining reduced-dose chemotherapy with immunotherapy. Two personalized constructs were designed: MEVC (303 aa), a “dual-TLR4 self-adjuvanting” architecture integrating two intrinsic TLR4 adjuvants (L7/L12, RS09) flanking seven epitopes (three MHC-I, three MHC-II/Th, one B-cell); and CRUISER (Chemokine-Recruited Universal Immune-Stimulating Epitope Reservoir), an mRNA-LNP construct encoding a 259-aa ORF that combines the chemokine XCL1 for cDC1 (XCR1⁺) recruitment, a patient-specific epitope cassette (SCRN1–CBL, MSH2, HDAC8, IL2RA/CD25, PADRE), and a T4 fibritin foldon enabling self-assembly into a soluble ~85 kDa trimer. Unlike autogene cevumeran (BNT122), which membrane-anchors antigens via MITD, CRUISER is secreted as a soluble trimer that actively recruits cDC1 through the XCL1–XCR1 axis - among the first trimeric chemokine–neoantigen mRNA vaccines reported for any hematologic malignancy. GROMACS confirmed structural stability of both constructs, and C-ImmSim showed both simultaneously activate humoral immunity (class-switching, durable B-cell memory), balanced Th1/Th2 helper responses, and cytotoxic T-cell responses with lasting memory, within a balanced cytokine profile and no evidence of chronic inflammation or immune tolerance.

Conclusion: This work integrates immortalized blast cell line establishment with whole-genome sequencing to enable subsequent in vitro and preclinical studies, while helping close the Southeast Asian representation gap in hematologic-cancer omics. The two multivalent constructs MEVC and CRUISER achieved structural stability and a favorable in silico immunogenicity profile, and are ready for experimental evaluation on the AL035 immortalized blast line in preclinical studies.

Dr. Loc Pham Thi is Head of the Department of Hematology and Blood Transfusion at Hai Phong University of Medicine and Pharmacy, Viet Nam, and a collaborative professor jointly appointed with Kanazawa University, Japan, where Dr. Pham earned a PhD in Medicine. Dr. Pham currently leads two government-funded projects: CRISPR/Cas9 editing on cancer patient samples and stem-cell and applied-immunology approaches for hybrid TAA/TSA and personalized leukemia vaccines; regenerative-medicine therapy for knee osteoarthritis patients (grant codes ĐT.YD.2023.966 and ĐT.YD.2025.978). Research interests center on hematologic malignancies, cancer genomics, and personalized cancer immunotherapy.