Design and development of a bispecific antibody targeting BAFF and IL-17 for systemic lupus erythematosus treatment

Xin, Cheng; Zhou, Jingming; Chen, Yumei; Liu, Yankai; Liu, Hongliang; Liang, Chao; Zhu, Xifang; Qi, Yanhua; Zhang, Gaiping; Wang, Aiping

doi:10.1186/s12915-025-02398-2

Methodology
Open access
Published: 29 September 2025

Design and development of a bispecific antibody targeting BAFF and IL-17 for systemic lupus erythematosus treatment

BMC Biology volume 23, Article number: 283 (2025) Cite this article

398 Accesses
1 Altmetric
Metrics details

Abstract

Background

This study aimed to develop and evaluate a bispecific single-chain variable fragment (bsscFv) targeting B-cell activating factor (BAFF) and interleukin-17 (IL-17) for the treatment of systemic lupus erythematosus (SLE).

Methods

The bsscFv was engineered by linking single-chain variable fragments (scFvs) specific for BAFF and IL-17 with a flexible peptide linker. It was expressed in E. coli BL21 and purified using affinity chromatography. Binding affinities to BAFF and IL-17 were assessed by enzyme-linked immunosorbent assay (ELISA). In vitro neutralization assays were conducted using Raji and HT-29 cell cultures. In vivo therapeutic efficacy was evaluated in an MRL/lpr mouse model of SLE, with 10 mice per group. Statistical significance was determined using a Student’s t-test for comparison of two groups.

Results

The bsscFv demonstrated strong binding to both BAFF and IL-17 in ELISA. In vitro, it inhibited BAFF-induced B-cell survival, proliferation, and immunoglobulin production, as well as IL-17-induced inflammatory cytokine secretion in HT-29 cells. In the MRL/lpr mouse model, bsscFv treatment significantly reduced autoantibody levels (p < 0.05), proteinuria, renal pathology, and cytokine expression in a dose-dependent manner compared to controls.

Conclusions

The bsscFv exhibited potent neutralizing activity in vitro and therapeutic efficacy in vivo, suggesting it as a promising bispecific therapeutic agent for the treatment of SLE. Further studies are warranted to explore its clinical potential.

Background

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by the production of autoantibodies, immune complex deposition, and multi-organ damage [1, 2]. The pathogenesis of SLE involves a complex interplay of genetic, environmental, and immunological factors, leading to the breakdown of self-tolerance and the development of autoimmunity [3, 4]. Despite advances in our understanding of SLE pathogenesis and the availability of various treatment options, many patients continue to experience significant morbidity and mortality, highlighting the need for novel therapeutic strategies [5].

B-cell activating factor (BAFF) and interleukin-17 (IL-17) are two key cytokines implicated in the pathogenesis of SLE [6,7,8]. BAFF, a member of the tumor necrosis factor (TNF) superfamily, plays a crucial role in B-cell survival, maturation, and autoantibody production [9, 10]. Elevated levels of BAFF have been observed in SLE patients and correlate with disease activity and autoantibody titers [11]. IL-17, produced by T helper 17 (Th17) cells, has been shown to contribute to SLE pathogenesis by promoting inflammation, tissue damage, and the recruitment of immune cells [12, 13]. Increased levels of IL-17 have been detected in the serum and target organs of SLE patients, suggesting its involvement in disease progression [14, 15].

Given the central roles of BAFF and IL-17 in SLE pathogenesis, targeting these cytokines represents a promising therapeutic approach. Monoclonal antibodies targeting BAFF, such as belimumab, have demonstrated clinical efficacy in SLE patients, leading to their approval for the treatment of SLE [16, 17]. Similarly, IL-17 inhibitors have shown potential in preclinical models of SLE and are currently being investigated in clinical trials [18, 19].

Bispecific antibodies, engineered to recognize two different antigens, have emerged as a powerful tool for the treatment of various diseases, including autoimmune disorders [20, 21]. By simultaneously targeting multiple disease-related pathways, bispecific antibodies offer the potential for enhanced therapeutic efficacy and reduced side effects compared to conventional monoclonal antibodies [22].

In this study, we aimed to develop and evaluate a novel bispecific single-chain variable fragment (bsscFv) targeting both BAFF and IL-17 for the treatment of SLE. We hypothesized that the simultaneous inhibition of these two key cytokines would result in enhanced therapeutic efficacy compared to targeting either cytokine alone. To test this hypothesis, we generated a panel of bsscFv using phage display technology and assessed their binding affinity, specificity, and in vitro functional activity. The lead bsscFv was then evaluated in a well-established murine model of SLE to determine its efficacy in reducing autoantibody production, kidney damage, and systemic inflammation. Our results demonstrate the potential of bsscFvs targeting BAFF and IL-17 as a novel therapeutic strategy for the treatment of SLE.

Results

Protein expression and purification

Recombinant human BAFF and IL-17 proteins were expressed in HEK293F cells and purified via Ni–NTA affinity chromatography. SDS-PAGE showed bands at the expected molecular weights of 18 kDa for BAFF and 16 kDa for IL-17 (Additional file 1: Fig. S1A). The purity of the proteins was > 95%, confirmed by densitometric analysis of Coomassie Blue-stained gels. Identity was further validated by Western blot using anti-His antibodies (Additional file 1: Fig. S1B).

Antibody titers and spleen cell isolation

Mice immunized with recombinant BAFF and IL-17 exhibited significantly higher antigen-specific antibody titers compared to pre-immune sera. Endpoint titers for anti-BAFF and anti-IL-17 antibodies reached 1:204,800 and 1:102,400, respectively, after the fourth immunization (Additional file 1: Fig. S1C and S1D). Spleens were harvested, and single-cell suspensions were prepared for antibody library construction, with cell viability exceeding 90% as assessed by trypan blue exclusion.

PCR of antibody chains and construction of scFv phage library

Total RNA was extracted from isolated spleen cells, and cDNA was synthesized using oligo(dT) primers. PCR amplification of the antibody light (VL) and heavy (VH) chain variable regions produced bands of approximately 400 bp and 500 bp, respectively (Additional file 1: Fig. S2A and S2B). The purified VL and VH fragments were cloned into the pCANTAB 5E vector, resulting in a phage library containing 10.5 × 10¹⁰ independent clones (Additional file 1: Table 2). PCR screening revealed > 93% of clones had inserts of the expected size (Additional file 1: Fig. S2C and S2D). Next-generation sequencing (NGS) analysis showed the library's VH and VK gene distribution matched natural germline frequencies (Additional file 1: Fig. S3A and S3B), with CDR3 sequence lengths following a normal distribution (Additional file 1: Fig. S3C) and random pairing of heavy and light chain variable regions (Additional file 1: Fig. S3D), indicating excellent sequence diversity for subsequent antibody screening.

Helper phage amplification and scFv library quality evaluation

TG1 plates infected with the helper phage were subjected to gradient dilution, and colony counts were used to calculate the titer of amplified M13K07 helper phage (Additional file 1: Table 3). Using a 10^–6 dilution, 211 colonies were counted, resulting in a titer of 5.27 × 10¹⁴ pfu/mL from a total of 20 mL. This titer meets the requirements for subsequent screening. Similarly, the amplified scFv phage library was evaluated by gradient dilution of TG1 plates infected with the phage, yielding 125/98 colonies at a 10^–3 dilution (Additional file 1: Table 4). The final titer of the phage library was 6.25 × 10¹²/4.9 × 10¹² pfu/mL, indicating sufficient quantity for further screening.

Phage display selection

The scFv library underwent five rounds of phage display selection against recombinant BAFF and IL-17 proteins (Additional file 1: Table 5). In each round, phages binding to the targets were eluted and amplified. After the fifth round, individual clones were screened using phage ELISA, and positive clones (OD450nm > 2) were sequenced (Additional file 1: Fig. S4). Two unique scFv clones, scFv1 and scFv2, were identified as specific binders to BAFF and IL-17, respectively.

Construction and expression of bsscFv

As shown in Fig. 1A, scFv1 and scFv2 sequences were fused via a flexible linker (G₄S)₂ to create the bsscFv construct. The bsscFv gene was cloned into a pET28a(+) vector and transfected into BL21(DE3) cells for protein expression (Additional file 1: Fig. S5A). The protein was purified using affinity chromatography from disrupted cell lysates. SDS-PAGE and Western blotting with anti-His antibodies confirmed the purified protein (Fig. 1B, C). To quantitatively assess the binding affinity of the bispecific antibody, we performed label-free kinetic measurements using the Gator Prime system. The determined kinetic parameters showed that bsscFv exhibited high-affinity binding to both BAFF and IL-17, with KD values in the low nanomolar range (Table 1). Importantly, pre-saturation of bsscFv with either BAFF or IL-17 did not significantly alter the binding kinetics to the second target, indicating independent, non-competitive engagement of both epitopes (Additional file 1: Fig. S5B, S5C). These results confirm that the bispecific antibody maintains dual specificity and high-affinity binding to each target independently, even in the presence of the other.

Table 1 Kinetic parameters of bsscFv and monospecific scFvs binding to BAFF and IL-17

Full size table

In vitro experiments

The neutralizing activity of bsscFv was assessed in Raji and HT-29 cells. BAFF promoted Raji cell proliferation (Fig. 2A), and IL-17 induced CXCL1 secretion in HT-29 cells (Fig. 2B). bsscFv inhibited BAFF-induced Raji cell proliferation and IL-17-mediated CXCL1 secretion. IL-17 did not affect bsscFv's inhibition of Raji cell proliferation (Fig. 2C), nor did BAFF affect its inhibition of CXCL1 secretion (Fig. 2D). The IC50 of bsscFv for Raji cell proliferation was 0.39 ± 0.04 nM, similar to scFv1 (0.43 ± 0.02 nM), and for HT-29 cells, it was 4.63 ± 0.35 nM, comparable to scFv2 (4.12 ± 0.19 nM). These results show that bsscFv blocks both BAFF and IL-17 effectively.

In vivo experiments

A single injection of scFv1/bsscFv significantly reduced CD19 transcription levels in mouse spleens (Fig. 2E), indicating effective BAFF neutralization. Similarly, scFv2/bsscFv injection significantly decreased CXCL1 levels in mice (Fig. 2F), demonstrating effective IL-17 antagonism.

PK/PD

The pharmacokinetic (PK) profile of bsscFv was assessed in mice following IV doses of 1, 5, or 10 mg/kg and SC doses of 5 mg/kg. The PK data showed non-linear behavior, with serum concentrations declining in a dose-dependent manner. The area under the curve (AUC_0-∞) increased disproportionately with higher doses, and the terminal half-life extended from 3 to 8 days, likely due to target-mediated elimination via BAFF. SC bioavailability was 93%, with peak concentrations observed 3 days post-injection. Binding assays confirmed bsscFv's functional integrity and no antidrug antibodies (Fig. 2G). Both BAFF and IL-17 levels in serum were modulated in a dose-dependent manner following bsscFv administration, reflecting the pharmacodynamic (PD) effects of the antibody (Fig. 2H, I). PD analysis showed a dose-dependent reduction in circulating B cells, with 50% ~ 60% inhibition at 2 months post-5 mg/kg injection (Fig. 2J), confirming BAFF neutralization and bsscFv’s biological activity.

Effect of bsscFv treatment on urinary protein and autoantibody levels in MRL/lpr mice

Mice were divided into five groups: BALB/c control (n = 5), MRL/lpr model (n = 5), and MRL/lpr model with low (1 mg/kg, n = 5), medium (5 mg/kg, n = 5), or high (10 mg/kg, n = 5) doses of bsscFv, administered intraperitoneally twice weekly for 8 weeks. As shown in Fig. 3A-H, urinary protein levels decreased significantly in the medium- and high-dose groups. BUN levels also showed a dose-dependent reduction. Anti-dsDNA antibody levels, serum creatinine (Scr), ANA, anti-snRNP/Sm antibodies, and SLE-IgG levels were lower in the bsscFv-treated groups. The Urine Protein/Creatinine Ratio was significantly reduced in the medium- and high-dose groups.

Influence of bsscFv treatment on cytokine levels and correlation with target distribution in MRL/lpr mice

As illustrated in Fig. 3I, expression levels of Il-2, Il-4, Il-6, Il-10, Ifnα, Tnfα and Cd83 in murine peripheral blood mononuclear cells were downregulated following bsscFv treatment. Figure 3J and K indicates that protein levels of IL-2, IL-4, IL-6, IL-10, IFN-α and TNF-α in kidney tissues were significantly reduced as a result of bsscFv treatment.

Histopathological improvements and reduced immune complex deposition in bsscFv-treated MRL/lpr mice

Figure 4A-C shows that HE, PAS, and Masson’s trichrome staining revealed improved renal pathology in the bsscFv-treated groups, with reduced glomerular and tubulointerstitial lesions. In HE staining, yellow arrows point to glomeruli, red arrows to renal tubules, and black arrows to interstitial areas. The MRL/lpr Control group exhibited tubular epithelial loosening, edema, and detachment. However, the treated group showed well-preserved, rounded tubular epithelial cells with orderly brush borders, confirming the treatment’s effectiveness. PAS and Masson’s trichrome staining further confirmed significant improvements in renal pathology in the treatment group. As shown in Fig. 4D and E, immune complex deposition in the kidneys was notably reduced in the bsscFv-treated groups, further supporting the therapeutic efficacy.

Transcriptomic and metabolomic analyses reveal therapeutic effects of bsscFv treatment in MRL/lpr mice

Transcriptomic and metabolomic analyses of kidneys from the MRL/lpr model (M) and bispecific antibody-treated group (T) showed that bsscFv targeted immune responses and metabolic pathways. GO enrichment analysis revealed upregulation of immune-related processes, such as responses to stimuli, suggesting improved immune defense and reduced autoimmune damage. Enriched genes related to adaptive immunity and regulation indicated reduced pathological immune responses. Cellular components like phagocytic vesicles and immunoglobulin complexes suggested increased phagocytosis and antibody-mediated immunity, potentially alleviating kidney damage (Fig. 5A). KEGG pathway analysis highlighted metabolic pathways, such as peroxisome, phagosome, and fatty acid degradation, indicating reduced oxidative stress and inflammation. Lipid metabolism regulation, particularly unsaturated fatty acid biosynthesis, suggests protection from lipid peroxidation and kidney damage (Fig. 5B). Metabolomic clustering revealed higher levels of metabolites like (4-Oxo-1,2,3-benzotriazin-3(4H)-yl) acetic acid and anti-inflammatory compounds (Beclomethasone, Flurandrenolide) in the treated group (Fig. 5C). KEGG heatmap analysis showed changes in nucleotide and lipid metabolism, suggesting that bsscFv reduced metabolic imbalances and kidney damage associated with lipid disorders (Fig. 5D).

Analysis of antigen–antibody binding sites

The three-dimensional structure of bsscFv was predicted using AlphaFold (Additional file 1: Fig. S6A). The structures of BAFF (PDB ID: 1KD7) and IL-17 (PDB ID: 4HR9) were preprocessed using Discovery Studio and visualized in PyMOL (Additional file 1: Fig. S6B and S6C). The interactions are illustrated in Additional file 1: Fig. S6D, where bsscFv is shown in green, BAFF in blue, and IL-17 in purple. Yellow dashed lines indicate hydrogen bonds, while red dashed lines represent hydrophobic interactions. The binding scores for bsscFv-BAFF and bsscFv-IL-17 are −295.89 and −288.00, respectively, with confidence scores of 94%, indicating strong binding stability. Key interactions include hydrogen bonds between BAFF’s MET236/THR239 and bsscFv’s TYR176/TYR103 (3.1 Å, 3.0 Å). For IL-17, ASN25 forms a hydrogen bond with bsscFv’s THR320 (2.7 Å), and ARG20 of IL-17 forms three hydrogen bonds with bsscFv’s TYR363, ASN365, and TYR367 (2.3 Å, 2.5 Å, 3.1 Å). In Additional file 1: Fig. S6E, "A" represents bsscFv and "B" represents BAFF, while Additional file 1: Fig. S6F shows "A" as bsscFv and "B" as IL-17, further confirming these binding interactions. These interactions are critical for the stable binding between the bsscFv, BAFF, and IL-17 proteins.

Survival analysis of mice during the experimental period

Kaplan–Meier survival analysis was conducted to evaluate the mortality of mice across all in vivo experimental groups, as shown in Supplementary Additional file 1: Fig. S7. Throughout the treatment period, all groups were monitored for survival, and no deaths were observed in any cohort that could be attributed to treatment-related toxicity or disease progression. Statistical analysis using the log-rank (Mantel–Cox) test revealed no significant differences in survival rates among the groups (P > 0.05). These findings indicate that administration of the bispecific antibody, across the tested doses and experimental conditions, did not adversely affect survival and was well tolerated in vivo.

Discussion

This study presents the development and evaluation of a bsscFv targeting BAFF and IL-17 for the treatment of SLE. The bsscFv was engineered by fusing scFvs specific to BAFF and IL-17 via a flexible linker, yielding low-nanomolar binding affinities for both targets, comparable to their parental antibodies.

In vitro assays demonstrated potent dual neutralization by the bsscFv, effectively inhibiting BAFF- and IL-17-mediated B-cell survival, proliferation, immunoglobulin production, and proinflammatory cytokine secretion by peripheral blood mononuclear cells. These findings underscore the dual capacity of bsscFv to disrupt two critical immunopathological axes in SLE: autoreactive B-cell activation and systemic inflammation.

In vivo, bsscFv treatment in the MRL/lpr murine model—recapitulating major features of human SLE—led to dose-dependent reductions in autoantibody titers, proteinuria, and glomerulonephritis. These effects were accompanied by a broad suppression of proinflammatory cytokines (IL-2, IL-4, IL-6, IL-10, IFN-α, and TNF-α) in both kidney and spleen tissues, reinforcing the therapeutic potential of dual-pathway blockade.

When compared to current therapeutic strategies, the bsscFv offers several potential advantages. For instance, belimumab, a monoclonal antibody targeting BAFF, has been approved for SLE but exhibits limited efficacy in patients with high disease activity or refractory manifestations. Similarly, IL-17-targeted therapies such as secukinumab have shown promise in autoimmune diseases like psoriasis and ankylosing spondylitis, but their role in SLE remains exploratory and is often limited by partial responses. Moreover, dual therapy combining BAFF and IL-17 inhibitors may enhance disease control but is associated with increased cost, complexity, potential for overlapping toxicities, and reduced patient adherence.

In contrast, the bsscFv format provides a streamlined and mechanistically synergistic approach, integrating dual specificity into a single molecular entity. This may result in more efficient immune modulation, favorable pharmacokinetics, reduced dosing frequency, and better patient compliance [23]. Furthermore, bispecific antibodies have been shown to induce cooperative effects that exceed those of monotherapies or their combinations, possibly by simultaneously engaging spatially or temporally linked immune mechanisms.

The favorable safety signals observed in preliminary non-human primate studies further support the clinical translatability of the bsscFv. Nonetheless, this study has limitations. The therapeutic efficacy was assessed in a single murine model, and the cellular selectivity of bsscFv remains incompletely characterized. While cytokine inhibition and disease amelioration were clearly demonstrated, the preferential targeting of autoreactive versus non-pathogenic lymphocyte subsets was not directly assessed.

Encouragingly, ongoing work (manuscript in preparation) suggests that bsscFv treatment selectively reduces pathogenic cytokine production and autoantibody levels, with minimal impact on total immunoglobulin levels and naïve lymphocyte populations. These data imply a degree of immunological precision, potentially arising from elevated expression of BAFF and IL-17 in activated immune niches.

To further address these critical questions, future studies should incorporate high-resolution profiling approaches, such as single-cell RNA sequencing or multi-parametric flow cytometry, to delineate bsscFv’s cellular targets in murine and human immune systems. Additionally, comprehensive preclinical studies evaluating long-term safety, efficacy, and immunogenicity are necessary before clinical translation.

In conclusion, our findings provide compelling evidence that bispecific targeting of BAFF and IL-17 offers a promising therapeutic approach for SLE by simultaneously modulating B-cell-driven autoimmunity and inflammatory cytokine networks. Compared to existing monotherapies and combination regimens, the bsscFv holds distinct advantages in molecular efficiency, breadth of action, and potential clinical convenience. Further mechanistic and translational investigations are warranted to establish its safety, specificity, and therapeutic durability in human SLE.

Conclusions

In this study, we successfully engineered and characterized a bsscFv antibody targeting BAFF and IL-17, two pivotal cytokines implicated in the pathogenesis of SLE. The bsscFv exhibited robust binding affinity to both targets with minimal cross-interference, and effectively neutralized BAFF- and IL-17-mediated biological activities in vitro. In vivo administration of bsscFv in MRL/lpr mice led to significant reductions in autoantibody levels, proteinuria, renal pathology, and inflammatory cytokine expression, accompanied by histopathological improvements and decreased immune complex deposition in kidney tissues. PK and PD analyses confirmed its favorable bioavailability, biological activity, and lack of observable toxicity. Multi-omics analyses further supported the therapeutic impact of bsscFv on immune regulation and metabolic rebalancing. Taken together, these results indicate that bsscFv is a promising bispecific therapeutic candidate for the treatment of SLE, meriting further preclinical and clinical development.

Methods

Vector construction and cell culture

The coding sequences of human BAFF (UniProt: Q9Y275, residues A134–L285) and IL-17A (UniProt: Q16552, residues G24–A155) were amplified by PCR and cloned into the pCDNA3.1(+) vector (Thermo Fisher). The plasmids were transfected into HEK 293 F cells using polyethylenimine (PEI, Polysciences, 1 mg/mL, pH 7.0) at a DNA:PEI ratio of 1:3. Transfected cells were cultured in FreeStyle™ 293 Expression Medium (Gibco) for 72 h at 37 °C, 5% CO₂ with shaking (120 rpm). Supernatants were harvested by centrifugation at 4000 × g for 15 min. The scFv variable regions targeting BAFF and IL-17 were cloned into pET-28a(+) (Novagen), with a flexible (G₄S)₂ linker, to construct bispecific scFv (bsscFv). Plasmids were transformed into E. coli BL21(DE3) (TransGen Biotech), cultured in LB medium with 50 μg/mL kanamycin, induced with 0.5 mM IPTG at 16 °C for 16 h when OD600 reached 0.6. Raji and HT-29 cells were cultured in RPMI-1640 and McCoy’s 5 A media (Gibco), respectively, supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (100 U/mL penicillin, 100 μg/mL streptomycin) at 37 °C in a 5% CO₂ incubator.

Protein purification and expression

Bacterial pellets were resuspended in lysis buffer (50 mM Tris–HCl, pH 8.0; 300 mM NaCl; 10 mM imidazole; 1 mM PMSF), sonicated on ice (10 s on/20 s off cycles for 10 min), and centrifuged at 12,000 × g for 30 min at 4°C. The supernatant was applied to a Ni–NTA agarose column (Qiagen) pre-equilibrated with the same buffer. After washing with wash buffer (50 mM Tris–HCl, 300 mM NaCl, 30 mM imidazole, pH 8.0), bound proteins were eluted with elution buffer (50 mM Tris–HCl, 300 mM NaCl, 250 mM imidazole, pH 8.0). Proteins were dialyzed against PBS (pH 7.4) and concentrated using Amicon Ultra-15 centrifugal filters (30 kDa cutoff, Millipore). Protein concentration was quantified by Bradford assay (Bio-Rad), and purity was confirmed via 12% SDS-PAGE.

Western blot

Proteins (10 μg/lane) were separated by SDS-PAGE and transferred to a 0.45 μm PVDF membrane (Millipore) using a semi-dry blotting system (Bio-Rad). Membranes were blocked with 5% non-fat milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature. Primary antibody: anti-His monoclonal antibody (1:5000, Abcam, ab18184), incubated overnight at 4°C. After washing, membranes were incubated with HRP-conjugated goat anti-mouse IgG (1:5000, Abcam, ab6789) for 1 h at room temperature. Signal was developed using the ECL Plus detection kit (Tanon).

Mouse immunization protocol and spleen cell isolation

Female BALB/c mice (6–8 weeks old) were purchased from the Zhengzhou University Experimental Animal Center and the Henan Province Experimental Animal Center. All animal experiments were conducted in accordance with the guidelines of the Animal Ethics Committee of Zhengzhou University, and the study protocol was approved by the committee (Approval No. ZZUIRB2021-141). Prior to immunization, mice were anesthetized using isoflurane inhalation (4% for induction, 2% for maintenance) to minimize discomfort. At the study endpoint, euthanasia was performed by CO₂ inhalation, in line with the AVMA Guidelines for the Euthanasia of Animals (2020). The animals were immunized with recombinant BAFF (50 μg) and IL-17 (50 μg) proteins in CFA on day 0, followed by booster immunizations on days 14 and 28 using IFA. On day 35, blood samples were collected for antibody titers. A final booster (50 μg BAFF and IL-17) was administered on day 42. Three days later, mice were euthanized, and spleens were harvested, disrupted, filtered, and centrifuged. Spleen cell suspensions, rather than purified B lymphocytes, were used in downstream assays to preserve the native immunological microenvironment and maintain physiological cell–cell interactions essential for cytokine-driven immune responses, including T cell help, antigen presentation, and cytokine crosstalk. This approach provides a more biologically relevant model for evaluating the activation and transcriptional responses of B cells within the splenic milieu. Cells were treated with RBC lysis buffer, resuspended in PBS, counted using trypan blue, and prepared for RNA extraction and cDNA synthesis (Fig. 6A).

mRNA extraction, reverse transcription, and qPCR

Total RNA was extracted using the FastPure® Cell/Tissue Total RNA Isolation Kit (Vazyme RC101), quantified with a NanoDrop 2000 spectrophotometer (Thermo). Reverse transcription was performed using the PrimeScript™ RT reagent Kit (Takara). qPCR was conducted using ChamQ SYBR qPCR Master Mix (Vazyme Q331) on a LightCycler 96 system (Roche). Gene expression levels were normalized to GAPDH, and relative changes calculated using the 2^−(ΔΔCt) method [24].

PCR amplification of antibody light and heavy chain variable regions

The antibody light (VL) and heavy (VH) chain variable regions were amplified from cDNA using PCR with degenerate primers (Additional file 1: Table 1) based on mouse immunoglobulin sequences. The PCR mixture (50 μL) included 1 × buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.5 μM primers, 1 U Taq polymerase (Takara), and 2 μL cDNA. PCR conditions: 95 °C for 5 min, 30 cycles (95°C for 30 s, 58 °C for 30 s, 72 °C for 1 min), final extension at 72 °C for 5 min. PCR products were purified (Tiangen kit) and analyzed by agarose gel electrophoresis.

Analysis of scFv insertion rate and antibody library sequence diversity

Insertion Rate: Forty-eight DH5α/pCANTAB 5E single colonies were selected for PCR amplification to evaluate the insertion rate of scFvs based on the presence or absence of PCR products. Antibody Sequence Diversity: Next-generation sequencing (NGS) was used to assess the diversity of the antibody library sequences.

M13K07 helper phage amplification and titer determination

M13K07 phage (Fenghui Biotech) was amplified in E. coli TG1 cells (Beijing Huayueyang Biotechnology) in 2 × YT medium with 50 μg/mL kanamycin. The culture was incubated at 37 °C until OD600 reached 0.5, then phage was added at MOI 20 and incubated for 1 h. Infected cells were grown overnight at 30°C. Phage was precipitated using 20% PEG/2.5 M NaCl, resuspended in PBS, and titered by infecting E. coli TG1 cells with serial dilutions and plating on LB agar with kanamycin.

Construction and quality assessment of scFv library

The scFv library was constructed by linking VL and VH sequences with a (G₄S)₄ linker via overlap extension PCR. The fragments were digested with BamHI and XhoI, ligated into pCANTAB 5E, and transformed into E. coli TG1 cells. Library size was estimated by colony count. Random clones were screened by PCR with pCANTAB 5E-specific primers and analyzed by gel electrophoresis to confirm full-length scFv inserts.