Targeting B-cell lymphoma surface protein with a novel peptide: Therapeutic potential and clinical implications

Abstract Background: B-cell lymphoma has significantly improved patient survival, however, approximately 30% of patients develop relapsed/refractory disease, which exhibits poor response to conventional therapies and unfavorable prognosis. To address this therapeutic gap, targeted therapy, aimed at enhancing treatment precision, holds promise for improving outcomes. This study employed phage display technology（PhD）to screen for peptides targeting B-cell lymphoma surface proteins and investigate their biological functions in lymphoma cells. We developed a novel phage display-based peptide-drug conjugate (PDC) strategy that utilizes tumor-specific peptides to deliver cytotoxic payloads (the pro-apoptotic KLA peptide), enabling precise tumor killing while minimizing off-target toxicity Methods: Through three rounds of biopanning against lymphoma cell lines using a 12-mer phage library. One hundred phage monoclonal clones were isolated, sequenced, and analyzed to identify two candidate peptides (C1 and C2). These were conjugated to cytotoxic peptide KLA to generate PDCs C1-KLA, C2-KLA, with scrambled-sequence NsG-KLA as control. Peptide specificity was validated via flow cytometry across multiple lymphoma cell lines. Functional assays were performed after conjugating C1 and C2 to the pro-apoptotic peptide KLA (C1-KLA/C2-KLA), with NsG-KLA. In vitro cytotoxicity and cell proliferation assays, cell viability was measured by CCK-8 assay, and proliferation was assessed using EdU incorporation. Cell apoptosis was analyzed by Annexin V-FITC/PI staining. In vivo tumor xenograft studies, a murine xenograft model was established by subcutaneously inoculating [TOLEDO] cells into [6weeks/nude mice] immunodeficient mice (n=[10]). Tumor growth was monitored by measuring tumor volume ([π/6 × width2 × length]). Results: C1 exhibited nanomolar affinity for lymphoma cells, with >90% target engagement at 20 μM with negligible cross-reactivity to normal B-cells (p<0.001). Synthetic peptides and their NsG controls were generated for downstream experiments. Encouragingly, in vitro cytotoxicity, apoptosis, proliferation assays, C1-KLA demonstrated significant pro-apoptotic, anti-proliferative, and cell growth-inhibitory effects. Conjugating to the KLA, C1-KLA induced caspase-3-dependent apoptosis (3.4-fold increase vs control, p<0.001) and inhibited proliferation. In murine xenograft model established with subcutaneous injection of human B-cell lymphoma cells （TOLEDO）in nude mice (n=10 per group), C1-KLA achieved objective responses in 100% of cases (at 120 mg/kg Q3D x 4 ), the tumor weight in the treatment group was significantly reduced compared to the control group (p<0.001), C1-KLA showed significant delay on tumor progression with no observable toxicity. Conclusion: Through comprehensive phage display screening and functional validation, we have identified C1 as a high-affinity targeting peptide that specifically recognizes a conserved epitope expressed in B-cell lymphoma cell lines (including DLBCL, FL subtypes). This peptide-drug conjugate (PDC) C1-KLA selectively targets a surface-exposed lymphoma-specific epitope, enabling tumor-restricted delivery of KLA. C1-KLA's small size (3.3 kDa) favors tissue penetration and scalable synthesis. These findings highlight C1-KLA as a novel and highly effective targeted therapeutic molecule specifically directed against lymphoma cell surface proteins, addressing the critical need for precision therapies in relapsed/refractory cases. This study establishes C1-KLA as a promising therapeutic candidate for B-cell lymphoma, with potential applicability to other CD19-negative malignancies. The PhD-to-PDC pipeline demonstrates broad applicability across hematologic malignancies expressing well-defined surface antigens, offering a streamlined and accelerated development pathway for precision-targeted immunotherapies.