Abstract Background Triple-negative breast cancer (TNBC) is an aggressive malignancy with limited therapeutic options. Immune checkpoint inhibitors targeting the programmed death-ligand 1 (PD-L1) pathway show restricted efficacy in TNBC, with response rates of only 5–10% as monotherapy. Interleukin-8 (IL-8/CXCL8) signaling promotes immunosuppression and mediates resistance to anti-PD-L1 therapy, necessitating combination approaches to overcome these limitations. However, the underlying mechanisms of enhanced efficacy from dual pathway targeting require further investigation. Methods We generated humanized mouse models by reconstituting immunodeficient mice with human PBMCs from five donors (n = 5 mice/group). MDA-MB-231 TNBC cells were implanted subcutaneously, and mice were treated with vehicle control, atezolizumab (anti-PD-L1), HuMax-IL8 (anti-IL-8), combination therapy, or a novel bispecific antibody BP2402 targeting both PD-L1 and IL-8. Antitumor activity was assessed alongside single-cell RNA sequencing of tumors and mechanistic analyses including immunofluorescence and Western blot. Results Combination therapy demonstrated significantly enhanced tumor growth inhibition compared to atezolizumab monotherapy in responsive donor models (51.28% vs. 39.13% for donor 3, p < 0.01; 44.01% vs. 6.57% for donor 4, p < 0.01). Single-cell RNA sequencing showed higher intratumoral T-cell fractions with combination therapy (donor 3: 80.5% vs. 26.7%; donor 4: 63.6% vs. 13.0% compared to control). BP2402 maintained high binding affinity for both IL-8 (KD = 2.132 nM) and PD-L1 (KD = 1.473 nM), and demonstrated superior antitumor efficacy compared to monotherapies (p < 0.001 vs. vehicle, p < 0.01 vs. individual antibodies). BP2402 treatment significantly reduced CXCL8 and VEGFA expression, suppressed JAK1/STAT1 signaling pathway activation, and upregulated pro-apoptotic proteins including FAS and BAX while effectively modulating T cell exhaustion markers PD-1 and TIM-3. Conclusions These results indicate that dual targeting of PD-L1 and IL-8 pathways represents a promising therapeutic strategy for TNBC. The bispecific antibody approach offers superior therapeutic potential by simultaneously modulating immune checkpoints, inflammatory signaling, and angiogenesis, effectively addressing resistance mechanisms. Additional preclinical optimization and clinical studies are required to fully assess the therapeutic potential of this novel immunotherapeutic approach. Keywords: Bispecific antibody, PD-L1, IL-8, Cancer immunotherapy, Tumor microenvironment, Triple-negative breast cancer Background Triple-negative breast cancer (TNBC) is recognized as a highly aggressive subtype of breast cancer, accounting for approximately 10–15% of all breast cancer cases [[60]1]. Characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), TNBC poses significant challenges due to its rapid progression, high metastatic potential, and limited treatment options [[61]2]. Patients with TNBC experience significantly poorer prognoses compared to those with other breast cancer subtypes, with five-year survival rates markedly lower than those of hormone receptor-positive breast cancers [[62]3]. While immune checkpoint inhibitors (ICIs), notably PD-1 and PD-L1 inhibitors, have improved overall survival in various advanced solid tumors, many patients still exhibit inadequate responses to these treatments. Immunosuppressive conditions within the tumor microenvironment (TME) hinder the effectiveness of ICIs, thereby reducing their clinical efficacy. Therefore, there is an urgent need for novel treatment combinations that enhance the TME, making it more conducive to effective immune checkpoint inhibition [[63]4]. Interleukin-8 (IL-8) has emerged as a crucial mediator of tumor progression and immune evasion across multiple cancer types, including TNBC [[64]5]. This proinflammatory chemokine is expressed by numerous solid tumors, including those of the brain, breast, cervical, colon, gastric, lung, melanoma, mesothelioma, ovarian, prostate, renal, and thyroid tissues, facilitating their growth, survival, and spread [[65]6]. Elevated serum levels of IL-8 have been associated with tumor progression and poor clinical outcomes in patients treated with PD-L1 inhibitors [[66]7, [67]8]. Specifically, high circulating IL-8 levels correlate with reduced clinical benefit from immune checkpoint inhibitors, suggesting that suppressing IL-8 may enhance treatment efficacy. Moreover, IL-8 plays a pivotal role in shaping the TME by facilitating the infiltration of neutrophils and myeloid-derived suppressor cells (MDSCs), collectively contributing to an immunosuppressive milieu that hinders T cell activity [[68]6, [69]9]. Importantly, IL-8 is closely linked to the vascular endothelial growth factor (VEGF) signaling pathway, promoting angiogenesis and epithelial-mesenchymal transition (EMT)—prominent processes that aid in metastasis [[70]10, [71]11]. Recent studies have suggested that targeting both PD-L1 and IL-8 might represent a novel therapeutic strategy [[72]12–[73]14]. By simultaneously inhibiting these immunosuppressive mechanisms, this approach aims to overcome the limitations of existing therapies. The proposed bispecific antibody that simultaneously targets PD-L1 and IL-8 not only aims to enhance anti-tumor immune responses in TNBC but also seeks to alter the TME to improve the clinical efficacy of immunotherapy. The primary objectives of this study are to develop and characterize this novel bispecific antibody, evaluate its potential to enhance anti-tumor immune responses, and investigate its efficacy in overcoming resistance to immunotherapy in TNBC. By addressing these critical immunosuppressive pathways, this strategy endeavors to tackle the significant challenges associated with TNBC immunotherapy and potentially improve clinical outcomes for patients suffering from this malignant disease. Materials and methods Mice Female C-NKG mice (6–8 weeks old) were purchased from Cyagen Biosciences (Suzhou) Co., Ltd. Animal care and experimental procedures were performed under SPF conditions. IVC mouse cages were used, with five mice per cage. All procedures involving mice were conducted in accordance with the guidelines outlined in the National Research Council's Guide for the Care and Use of Laboratory Animals, as well as the Institutional Animal Care and Use Committee (IACUC). The procedures also followed the guidelines provided by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Animals were housed in a temperature and humidity-controlled room with a 12-h light/dark cycle, and were provided with standard laboratory water and food. In vivo experiments MDA-MB-231 cells were cultured as a monolayer in Leibovitz’s L-15 medium supplemented with 15% fetal bovine serum (FBS; Gibco) and 1% antibiotic–antimycotic solution at 37 °C in a CO[2]-free incubator. When the cells reached 80–90% confluence, they were harvested and resuspended in serum-free L-15 medium at a concentration of 1 × 10^8 cells/mL. The cell suspension was then mixed with an equal volume of Matrigel® (Corning) to prepare the inoculum. Each mouse was subcutaneously injected with 0.2 mL of the cell/Matrigel® mixture into the right scapular region. Three days prior to tumor cell inoculation, human peripheral blood mononuclear cells (hPBMCs) from five donors were thawed, collected by centrifugation, and passed through a 70-μm cell strainer. The cells were then resuspended in serum-free RPMI-1640 medium and adjusted to a final concentration of 2.5 × 10⁷ cells/mL. Each mouse received 0.2 mL of the hPBMC suspension via tail vein injection. Upon reaching an average tumor volume of approximately 120 mm^3, the animals were stratified into randomized groups based on tumor volume, ensuring a variance within each group of less than 10% of the mean. Animal tumor volumes were measured thrice weekly by experimenters who were unaware of the specific agents administered. Tumor length and width were measured using digital calipers, and tumor volume was estimated from these measurements. The tumor volume was calculated using the following formula: tumor volume (mm^3) = [(width)2 × length]/2. Human donor characteristics In this study, PBMCs from five donors were utilized. The PBMCs were obtained from healthy donors and patients recruited at Shanghai Zhaxin Hospital of Traditional Chinese and Western Medicine. All PBMC collection and screening procedures were conducted under the approval of the Ethics Committee of Shanghai Zhaxin Hospital of Traditional Chinese and Western Medicine (approval number: XF0102232W) and in full compliance with the Declaration of Helsinki and medical ethics guidelines. All donors met the established inclusion criteria and were screened according to standard clinical protocols. All five donors were healthy males aged 20–30 years. Donor 3 detailed HLA typings are as follows: HLA-A: 33:03, 66:01; HLA-B: 41:XX, 58:01; and HLA-C: 03:02, 17:03. Similarly, Donor-4, HLA typings include HLA-A: 23:01, 34:02; HLA-B: 15:17, 53:01; and HLA-C: 04:01, 05:01. Both donors provided informed consent in accordance with ethical guidelines. The compatibility of their HLA types with the MDA-MB-231 cell line was evaluated, and it was noted that while Donor-3 may have a partial match, no complete matches were identified for either donor's HLA types with the MDA-MB-231 cells. These comprehensive details about the donors enhance the transparency and rigor of our study. Hematoxylin and Eosin (H&E) staining Tissue samples were fixed in 10% neutral buffered formalin for at least 24 h, processed through graded ethanol and xylene, and embedded in paraffin. Sections (4 μm) were cut, placed on glass slides, and dried. Standard H&E staining was performed: slides were dewaxed, rehydrated, stained with hematoxylin and eosin, dehydrated, cleared, and coverslipped. Histological analysis was conducted under a light microscope. Single-cell sequencing Single-cell preparation, library synthesis, RNA sequencing, and data analysis were completed by Gene Denovo (Guangzhou, China) using 10 × Genomics Chromium Single Cell Controller. Fresh tumor tissues were dissociated with Gentle MACS Dissociator (Miltenyi Biotec) according to the manufacturer’s instructions. Next, single cells were counted using an automated cell counter and adjusted to a concentration of 1000 cells/µL. After initial quality control, cellular suspensions were loaded on a 10 × Genomics GemCode Single-cell instrument that generates single-cell Gel Beads-In-Emulsions. The cells were lysed, and RNA was reverse transcribed into cDNA. Libraries were generated from the cDNA with Chromium Next GEM Single Cell 3′ Reagent Kit V.3.1. Finally, the cDNA libraries were sequenced using the Nova 6000 sequencing system (Illumina). 10 × Genomics Cell Ranger software (version 3.1.0) was used to convert raw BCL files to FASTQ files, alignment and counts quantification. Subsequent data analysis, including principal component analysis and t-distributed stochastic neighbor embedding (t-SNE analysis), was achieved using the Seurat software. Seurat embed cells in a shared-nearest neighbor graph, with edges drawn between cells based on similar gene expression patterns. Unsupervised density-based clustering by density-based spatial clustering of applications with noise (DBSCAN) clustering algorithm on the t-SNE analysis was performed to separate tumor cells into the distinct homogenous groups. Public dataset analysis To validate our preclinical findings, we analyzed scRNA-seq data from TNBC patients treated with anti-PD-1 immunotherapy ([74]GSE169246) [[75]15]. Among 10 patients, we selected three with highest and three with lowest intratumoral IL-8 gene expression for comparative analysis. Data processing and cell type annotation were performed using Seurat (v5.3.0) in R (v4.5.1). Major cell types and T cell subtypes were identified using canonical immune cell markers as described in references [[76]12, [77]15].