A Cell-Penetrating Peptide Improves Anti-HER2 Single-Chain Variable Fragment Internalization and Antitumor Activity against HER2-Positive Breast Cancer In Vitro and In Vivo

Cell-penetrating peptides (CPPs) are invaluable tools for delivering various substances into cells by crossing biological membranes. However, the effects of cell-penetrating peptide fusion proteins on the biological activity of antibodies remain to be fully understood. Here, we engineered a recombinant protein, LP-scFv, which combines the single-chain variable region of anti-human epidermal growth factor receptor-2 with a novel and non-oxic cell-penetrating peptide as a leader peptide. The introduction of this leader peptide led to a more than twofold increase in the internalization efficiency of the single-chain antibody, as confirmed using microscopic analysis and flow cytometry. The effects of the single-chain antibodies and LP-scFv on cell viability were evaluated using the MTT assay. Both the single-chain antibodies and LP-scFv reduced the viability of BT474 and NCI-N87 cells in a dose-dependent manner while exhibiting minimal toxicity towards MCF-7 and MCF-10A cells. Further investigation into LP-scFv’s mechanism revealed that the induced leader peptide does not alter the MAPK-ERK1/2 and PI3K/AKT pathways of single-chain antibodies. An enhanced antitumor activity was also confirmed in an NCI-N87 tumor xenograft model in mice with a reduction of 45.2% in tumor growth inhibition (vs. 23.1% for scFv) with a 50 mg/kg dose after orthotopic injection administration, which was equivalent to that of trastuzumab (vs. 55.7% for trastuzumab). Overall, these results indicate that LP-scFv exhibits significant permeation activity in HER2-positive cells to enhance the intracellular dose effect on antitumor activity in vitro and in vivo. This research lays the foundation for designing novel antibody-based therapies for cancer.


Introduction
Breast cancer, a clinically and molecularly heterogeneous malignancy, is a leading cause of cancer-associated mortality in women.In 2020, it accounted for 2.26 million new cases globally, representing 11.7% of all new cancer cases [1].Clinical studies have identified the human epidermal growth factor receptor 2 (HER2) as a crucial target in HER2-positive metastatic breast cancer therapy [2].HER2-targeted therapies, particularly antibody-related drugs, have significantly enhanced the prognosis of HER2-positive breast cancer [3].However, HER2-positive breast cancer patients often exhibit high primary or acquired drug resistance at both primary and metastatic sites [4,5].This resistance partly stems from the characteristics of monoclonal antibody drugs, a large molecular weight

Construction, Expression, and Purification of Recombinant scFv and LP-scFv
The purification of scFv, LP-scFv, scFv-EGFP, and LP-scFv-EGFP proteins was confirmed via SDS-PAGE and Western blotting.The results demonstrated that these recombinant proteins exhibited distinct bands, indicating a high purity (>90%), with molecular weights of approximately 31 kDa, 35 kDa, 55 kDa, and 58 kDa, respectively (Supplementary Materials Figure S1).
The binding affinities of the scFv and LP-scFv to HER2 were measured using labelfree biolayer interferometry (BLI) [28,29], as shown in Figure S2.Table 1 presents the calculated association (k on ) and dissociation (k off ) rate constants, along with the equilibrium dissociation constants (K D = k off /k on ).These values indicate comparable kinetic behaviors for both the scFv and LP-scFv in their interaction with HER2, implying that LP fusion did not compromise antigen-binding capabilities.

ScFv and LP-scFv Binding and Internalization
LP-EGFP was initially expressed in E. coli and purified using nickel affinity chromatography.This process yielded a distinct ~31 kDa band in both SDS-PAGE and Western blotting (Figure S1).Following a 24 h incubation of BT474 (a human breast ductal carcinoma cell line), NCI-N87 (a human gastric cancer cell line), MCF-7 (a human breast cancer cell line), MCF-10A (a human normal breast epithelial cell line), and human hepatic stellate LX2 cells with 500 nM LP-EGFP, flow cytometry was used to measure the fluorescence intensity in each cell line.This analysis aimed to assess the impact of LP coupling on the internalization of the macromolecular EGFP. Figure S3 demonstrated that, compared to EGFP alone, LP-EGFP exhibited a significantly improved internalization efficiency.This finding suggests that LP enhanced the internalization of the EGFP protein across different cell types without selectivity.
Internalization of cell-surface-bound scFv and LP-scFv fragments was assessed using established methods [30,31].As shown in Figure 1a, scFv-EGFP and LP-scFv-EGFP were specifically bound to HER2-positive BT474 cells at 4 • C, indicated by pronounced membrane fluorescence.Cells were then washed at 4 • C to remove unbound proteins, after which cells were incubated for 2 h at 37 • C; increased cytoplasmic fluorescence demonstrated their internalization.Confocal microscopy (Figure 1b) confirmed the binding and internalization of both scFv-EGFP and LP-scFv-EGFP in HER2-positive BT474 cells, with no significant fluorescence observed in HER2-negative MCF-7 cells.EGFP alone showed neither binding nor internalization in BT474 and MCF-7 cells.Flow cytometry was used to quantify the internalization efficacy of scFv-EGFP and LP-scFv-EGFP by measuring the fluorescence shifts on the cell surface relative to control cells (Figure 1c).The results showed that LP-scFv-EGFP internalization (41.2% ± 1.0%) was about twice that of scFv-EGFP (20.4% ± 2.4%), indicating that the LP significantly enhanced scFv-EGFP internalization in BT474 cells (Figure 1d).To confirm the endocytosis of scFv-EGFP and LP-scFv-EGFP into BT474 cells, threedimensional (3D) models were created.BT474 cells, treated with scFv-EGFP or LP-scFv-EGFP and stained with Hoechst 33342 and far-infrared DiD, were analyzed using confocal microscopy.Both scFv-EGFP (Figure 2a(i)) and LP-scFv-EGFP (Figure 2b(i)) were observed near the nucleus (blue), with LP-scFv-EGFP showing a more pronounced distribution.Three-dimensional sections of the BT474 cells showed that a part of scFv-EGFP or LP-scFv-EGFP (green; cyan arrow) was embedded in the cell membrane (red), indicating that this part of the protein bound to the HER2 antigen on the cell membrane and had not been endocytosed into the cell.The other part of the scFv-EGFP or LP-scFv-EGFP protein (green; yellow arrow) was apart from the cell membrane (red) and had access to the nucleus (blue), which indicated that this part of the scFv-EGFP or LP-scFv-EGFP protein had entered the cell (Figure 2a(ii),b(ii)).More importantly, most LP-scFv-EGFP (green; yellow arrow) was between the cell membrane (red) and the nucleus (blue), which indicated that most of the LP-scFv-EGFP protein was endocytosed into the cell.Quantitative fluorescence analysis of BT474 cells (Figure 2a(iii),b(iii)) showed 44.2% overlap of scFv-EGFP with the cell membrane (red), and 21.5% for LP-scFv-EGFP, indicating internalization of these proteins, particularly LP-scFv-EGFP.Laser confocal microscopy results suggest that coupling an LP with the scFv maintained specificity and improved scFv internalization.

Cytotoxicity and Apoptosis Induction
Experiments combining confocal microscopy and flow cytometry demonstrated that LP−scFv had no effect on the specific binding of the scFv to the HER2 antigen and increased the internalization efficiency of the scFv by about twofold.Subsequently, the impact of scFv and LP−scFv on cell viability was assessed using MTT assays and flow cytometry.Initially, the LP peptide, synthesized commercially, was co−cultured with BT474, NCI−N87, MCF−7, and MCF−10A cells at varying concentrations (0, 1, 5, 10, 25, 50 μg/mL) for 72 h, showing no significant effect on cell viability (Figure S4).Furthermore, the effect of the scFv and LP−scFv on the viability of MCF−10A, BT474, NCI−N87, and MCF−7 cells was evaluated, and the result indicated that both scFv and LP−scFv proteins were able to impair BT474 and NCI−N87 cell viability in a dose−dependent fashion, with LP−scFv exhibiting a superior anti-proliferative activity relative to the scFv (Figure 3a).In contrast, neither fragment could induce significant toxicity in MCF−7 and MCF−10A cells.To confirm apoptosis induction by scFv and LP−scFv treatments, flow cytometry was used to measure early (Q2; Annexin V + /PI − ) and late (Q3; Annexin V + /PI + ) apoptotic cells in BT474 and MCF−7 cells.Results showed that before treatment, most BT474 cells were alive (92.5% ± 0.5%).A total of 38.9% ± 1.6% of cells were apoptotic following LP−scFv treatment, compared to 17.3% ± 2.3% after scFv treatment, indicating LP-scFv's higher efficacy in inducing apoptosis.In contrast, both the scFv and LP−scFv induced minimal apoptosis in MCF−7 cells, with rates of 5.2% ± 0.9% and 6.1% ± 0.7%, respectively (Figure 3b,c).

Cytotoxicity and Apoptosis Induction
Experiments combining confocal microscopy and flow cytometry demonstrated that LP-scFv had no effect on the specific binding of the scFv to the HER2 antigen and increased the internalization efficiency of the scFv by about twofold.Subsequently, the impact of scFv and LP-scFv on cell viability was assessed using MTT assays and flow cytometry.Initially, the LP peptide, synthesized commercially, was co-cultured with BT474, NCI-N87, MCF-7, and MCF-10A cells at varying concentrations (0, 1, 5, 10, 25, 50 µg/mL) for 72 h, showing no significant effect on cell viability (Figure S4).Furthermore, the effect of the scFv and LP-scFv on the viability of MCF-10A, BT474, NCI-N87, and MCF-7 cells was evaluated, and the result indicated that both scFv and LP-scFv proteins were able to impair BT474 and NCI-N87 cell viability in a dose-dependent fashion, with LP-scFv exhibiting a superior anti-proliferative activity relative to the scFv (Figure 3a).In contrast, neither fragment could induce significant toxicity in MCF-7 and MCF-10A cells.To confirm apoptosis induction by scFv and LP-scFv treatments, flow cytometry was used to measure early (Q2; Annexin V + /PI − ) and late (Q3; Annexin V + /PI + ) apoptotic cells in BT474 and MCF-7 cells.Results showed that before treatment, most BT474 cells were alive (92.5% ± 0.5%).A total of 38.9% ± 1.6% of cells were apoptotic following LP-scFv treatment, compared to 17.3% ± 2.3% after scFv treatment, indicating LP-scFv's higher efficacy in inducing apoptosis.In contrast, both the scFv and LP-scFv induced minimal apoptosis in MCF-7 cells, with rates of 5.2% ± 0.9% and 6.1% ± 0.7%, respectively (Figure 3b,c).To confirm that LP−scFv enhances scFv's internalization and subsequent anticancer activity, BT474 cells were transiently transfected with pcDNA3.1−scFv−EGFP,and cell viability was assessed.BT474 cells transfected with pcDNA3.1−scFv−EGFPshowed significantly reduced viability compared to those transfected with pcDNA3.1−EGFP(Figure 4a−c), suggesting that the scFv inhibited cell proliferation post entry.Previous studies have reported that HER2 is expressed not only on the cell membrane but also in the nucleus and cytoplasm [32,33].To confirm scFv's interaction with cytoplasmic HER2 post transfection, thereby regulating epithelial cell proliferation and survival, HER2 protein levels in the membrane and cytoplasmic fractions of BT474 and MCF−7 tumor cell lines were measured, revealing full-length HER2 (185 kDa) in both the cell membrane and cytoplasmic fractions from BT474 cells, whereas only minimal membrane HER2 was detectable in MCF−7 cells (Figure S5).Therefore, LP−scFv may enhance the anti-proliferative effect of the scFv by increasing its intracellular dose through improved internalization.To confirm that LP-scFv enhances scFv's internalization and subsequent anticancer activity, BT474 cells were transiently transfected with pcDNA3.1-scFv-EGFP,and cell viability was assessed.BT474 cells transfected with pcDNA3.1-scFv-EGFPshowed significantly reduced viability compared to those transfected with pcDNA3.1-EGFP(Figure 4a-c), suggesting that the scFv inhibited cell proliferation post entry.Previous studies have reported that HER2 is expressed not only on the cell membrane but also in the nucleus and cytoplasm [32,33].To confirm scFv's interaction with cytoplasmic HER2 post transfection, thereby regulating epithelial cell proliferation and survival, HER2 protein levels in the membrane and cytoplasmic fractions of BT474 and MCF-7 tumor cell lines were measured, revealing full-length HER2 (185 kDa) in both the cell membrane and cytoplasmic fractions from BT474 cells, whereas only minimal membrane HER2 was detectable in MCF-7 cells (Figure S5).Therefore, LP-scFv may enhance the anti-proliferative effect of the scFv by increasing its intracellular dose through improved internalization.

Mechanism of LP-scFv and scFv Inhibiting the Proliferation of HER2-Positive BT474 Cells
Monoclonal antibodies targeting the HER2 receptor, including trastuzumab (Herceptin) and pertuzumab (Perjeta), inhibit downstream HER2 tyrosine kinase signaling via the MAPK-ERK1/2 and PI3K/AKT pathways, resulting in cell apoptosis and proliferation arrest [34,35].Considering that LP induction promoted scFv internalization and antitumor activity in HER2-positive cells in vitro, the mechanisms were further analyzed through the MAPK-ERK1/2 and PI3K/AKT pathways.The expression levels of apoptosis-related proteins, such as Caspase-3, cleaved Caspase-3, Bax, and Bcl2, were also investigated via Western blotting.The results indicated that BT474 cells treated with the scFv and LP-scFv exhibited significantly increased expressions of Bax and cleaved Caspase-3 and a significant decrease in Bcl2 levels compared to the control group.Notably, these effects were more pronounced with LP-scFv than with the scFv alone (Figure 5a-f).BT474 cells treated with the scFv and LP-scFv showed significant reductions in phosphorylated ERK1/2 (p-ERK1/2) and AKT (p-AKT) levels, with LP-scFv having a stronger effect than the scFv.In line with the flow cytometry results, neither the scFv nor LP-scFv induced apoptotic death or significant changes in AKT or ERK phosphorylation in MCF-7 cells (Figure 5g).These findings indicate that LP-scFv enhances the cytotoxic activity and apoptosis of the scFv in BT474 cells through the MAPK-ERK1/2 and PI3K/AKT pathways.Collectively, LP introduction promotes scFv internalization without altering its mechanism of action, improving the scFv's anticancer activity in HER2-positive BT474 cells by increasing its intracellular dosage.

Detection of scFv and LP-scFv's Antitumor Activity In Vivo
HER2 is a therapeutic target in both breast cancer and certain gastric tumors [36].The HER2-positive gastric cancer cell line, NCI-N87, serves as an effective model for HER2-targeting therapy in vivo [37,38].To assess LP-scFv's in vivo antitumor activity, mice bearing NCI-N87 tumor xenografts were used as models (Figure 6a). Figure 6b,c show that subcutaneous injections of trastuzumab, the scFv, or LP-scFv every other day significantly reduced the NCI-N87 tumor weight and size compared to the PBS-treated control group.Tumor growth inhibition (TGI%) was 55.7% for trastuzumab, 23.1% for scFv, and 45.2% for LP-scFv.No body weight loss was observed in any murine treatment group (Figure 6d).In the trastuzumab, scFv, and LP-scFv groups, tumor weights decreased by 68.1%, 31.8%, and 61.4%, respectively, compared to the control group (Figure 6e).These results indicate that LP-scFv significantly improved scFv's TGI rate.While LP-scFv's tumor suppression was not greater than that of trastuzumab, the difference was not statistically significant.

Detection of scFv and LP-scFv's Antitumor Activity In Vivo
HER2 is a therapeutic target in both breast cancer and certain gastric tumors [36].The HER2−positive gastric cancer cell line, NCI−N87, serves as an effective model for HER2targeting therapy in vivo [37,38].To assess LP−scFv's in vivo antitumor activity, mice bearing NCI−N87 tumor xenografts were used as models (Figure 6a). Figure 6b,c show that subcutaneous injections of trastuzumab, the scFv, or LP−scFv every other day significantly reduced the NCI−N87 tumor weight and size compared to the PBS−treated control group.Tumor growth inhibition (TGI%) was 55.7% for trastuzumab, 23.1% for scFv, and 45.2% for LP−scFv.No body weight loss was observed in any murine treatment group (Figure 6d).In the trastuzumab, scFv, and LP−scFv groups, tumor weights decreased by Ki-67, a nuclear DNA-binding protein associated with the cell cycle, is widely utilized as a marker of cell proliferation [39].Furthermore, the TUNEL assay is employed to evaluate apoptosis by labeling the exposed termini of DNA and visualizing nuclei with fragmented DNA [40].Ki-67 expression was reduced in the trastuzumab, scFv, and LP-scFv groups compared to the control group.TUNEL staining assays demonstrated that trastuzumab, scFv, and LP-scFv effectively induced cellular apoptosis (Figure 6f-h).LP-scFv inhibited tumor cell proliferation and enhanced apoptosis more significantly compared to scFv, aligning with our in vitro findings.
68.1%, 31.8%, and 61.4%, respectively, compared to the control group (Figure 6e).These results indicate that LP−scFv significantly improved scFv's TGI rate.While LP−scFv's tumor suppression was not greater than that of trastuzumab, the difference was not statistically significant.Ki−67, a nuclear DNA−binding protein associated with the cell cycle, is widely utilized as a marker of cell proliferation [39].Furthermore, the TUNEL assay is employed to evaluate apoptosis by labeling the exposed termini of DNA and visualizing nuclei with fragmented DNA [40].Ki−67 expression was reduced in the trastuzumab, scFv, and LP−scFv groups compared to the control group.TUNEL staining assays demonstrated that trastuzumab, scFv, and LP−scFv effectively induced cellular apoptosis (Figure 6f

Safety Evaluation of scFv and LP-scFv In Vivo
Following the animal experiments, major organs such as the heart, liver, spleen, stomach, and kidneys were collected for histopathological evaluation using hematoxylin and eosin (H&E) staining.Additionally, serum samples from the control and experimental groups were analyzed for liver and kidney function tests.Figure 7a indicates no significant morphological differences in the trastuzumab, scFv, and LP-scFv groups compared to the control group.Similarly, Figure 7b

Expression and Purification of LP−EGFP, scFv, and LP−scFv
The LP−EGFP, scFv, and LP−scFv genes were cloned between the BamHI and NdeI sites in the pET28a (+) vector with appropriate primers (Table S1).Competent E. coli BL21 (DE3) (Tiangen, Beijing, China) cells were transformed using these plasmids, after which cells were collected via centrifugation at 6500 rpm for 10 min at 4 °C.The cell pellets were lysed, supernatants were then loaded onto a Ni−NTA agarose column (GE Healthcare Life Sciences, Uppsala, Sweden), and bound protein was eluted with different concentrations of imidazole.SDS-PAGE and Western blotting were used to confirm expression and purity.Proteins were then dialyzed against PBS, concentrated to 0.5 mg/mL, and stored at −80 °C.

Assessment of scFv and LP−scFv HER2 Affinity
The affinity of the scFv and LP−scFv for HER2 was assessed using biolayer interferometry (BLI) with a ForteBio Octet RED96 system (Pall ForteBio, Fremont, CA, USA).For the analysis, both antibodies and the HER2 antigen were diluted in PBST buffer.Anti−human IgG Fc (AHC) biosensors were equilibrated in PBST for 10 min and then washed in PBST for 1 min to

Expression and Purification of LP-EGFP, scFv, and LP-scFv
The LP-EGFP, scFv, and LP-scFv genes were cloned between the BamHI and NdeI sites in the pET28a (+) vector with appropriate primers (Table S1).Competent E. coli BL21 (DE3) (Tiangen, Beijing, China) cells were transformed using these plasmids, after which cells were collected via centrifugation at 6500 rpm for 10 min at 4 • C. The cell pellets were lysed, supernatants were then loaded onto a Ni-NTA agarose column (GE Healthcare Life Sciences, Uppsala, Sweden), and bound protein was eluted with different concentrations of imidazole.SDS-PAGE and Western blotting were used to confirm expression and purity.Proteins were then dialyzed against PBS, concentrated to 0.5 mg/mL, and stored at −80 • C.

Assessment of scFv and LP-scFv HER2 Affinity
The affinity of the scFv and LP-scFv for HER2 was assessed using biolayer interferometry (BLI) with a ForteBio Octet RED96 system (Pall ForteBio, Fremont, CA, USA).For the analysis, both antibodies and the HER2 antigen were diluted in PBST buffer.Anti-human IgG Fc (AHC) biosensors were equilibrated in PBST for 10 min and then washed in PBST for 1 min to establish a baseline.Subsequently, recombinant human HER2 antigen (10 µg/mL, ab168896, Abcam, Cambridge, UK) was applied to the sensors for 100 s.The sensors were rinsed in a kinetics buffer for 1 min before being immersed in solutions with varying concentrations (100, 200, 300 nM) of the scFv or LP-scFv for a 160 s association step.The biosensors were then placed back into the assay buffer for a 160 s dissociation step.The resulting data were analyzed using a 1:1 Langmuir binding model with ForteBio 9.0 data analysis software.

Binding and Internalization Analysis
A total of 5 × 10 3 cells were seeded into 35 mm glass culture dishes (NEST, Beijing, China) for 24 h, after which they were treated with Hoechst 33342 (2 µg/mL; Invitrogen, Waltham, MA, USA) for 30 min at 37 • C. Cells were then incubated with either scFv-EGFP or LP-scFv-EGFP (30 µg) for 30 min at 4 • C, followed by washing with ice-cold PBS to remove unbound substances.After washing, the cells were incubated at 37 • C for 2 h to facilitate internalization.Subsequently, cells were examined using confocal laser scanning microscopy (600× magnification; Leica Microsystems, Wetzlar, Germany).BT474 cells treated with scFv-EGFP and LP-scFv-EGFP at 4 • C for 30 min were subsequently incubated at 37 • C for 2 h.Hoechst 33342 and DiD far-infrared were used to stain the nucleus and cell membrane, respectively.Imaging was performed using a laser confocal microscope equipped with a 63× (NA 1.46, Oil) objective lens and argon lasers (405 nm, 488 nm, and 650 nm).The image acquisition parameters were set as follows: x: 90 µm, y: 90 µm, z-axis step length: 0.54 µm, image size: 1024 × 1024.A total of 40 cell images were captured and stacked to create 3D cell images.
The binding and internalization of these fragments were further analyzed via flow cytometry, with the cells being processed as above.FlowJo vX software was used to determine the geometric mean fluorescence intensity for each sample, and the degree of internalization for these proteins was calculated based on the following formula [30,31]:

Measurement of Cell Apoptosis
BT474 and MCF-7 cells were seeded in 6-well plates and incubated overnight.Subsequently, the cells were treated with 500 nM of the scFv or LP-scFv for 72 h.Untreated cells served as controls.For the apoptosis analysis, a Cell Apoptosis Kit containing Annexin V AF488 and Propidium Iodide (PI) (Invitrogen, USA) was employed, with flow cytometry used to assess cell apoptotic death.

Cell Transfection
The pComb3HSS-scFv plasmid was utilized for the amplification of HER2 scFv, employing the primers detailed in Table S2.Subsequently, scFv genes were cloned between the AfIII and AgeI sites of the pcDNA3.1 (+)-EGFP vector.For transfection, 10,000 BT474 or MCF-7 cells were seeded per well in a 96-well plate and allowed to adhere overnight.A solution of 0.1 µg of plasmid DNA in 10 µL of medium was prepared and briefly vortexed.Separately, 0.4 µL of GenJet reagent (SL100489, SignaGen Laboratories, Frederick, MD, USA) was mixed with an additional 10 µL of media for each well.These solutions were then gently combined through pipetting and left to rest for 15 min.The mixture was then added to the cells in the 96 well plates.After 4-6 h of incubation, the mixture was replaced with fresh growth media.Cells were further incubated at 37 • C for 48 h.Post incubation, green fluorescence was observed using a fluorescence microscope to assess the transfection efficiency.

Assessment of Antitumor Effect in Xenograft Model Mice
The effect of the scFv and LP-scFv on tumor growth was assessed using tumor xenograft model mice.All animal procedures and studies were approved by the Institutional Animal Care and Use Committee of South China University of Technology (Guangzhou, China, Ref: 20180009, Date: 18 December 2019).Female BALB/c nude mice (3-4 weeks old, 16-18 g) were obtained from Hunan Slack Jingda Laboratory Animal Co., Ltd.(Changsha, China) and housed in a pathogen-free environment.The dorsal flank of each mouse was subcutaneously inoculated with 5 × 10 6 NCI-N87 cells.When tumor volumes reached 100-150 mm 3 , the nude mice were randomly divided into four groups for subcutaneous treatment [42][43][44], approximately 1 cm away from the tumor site.Details of the animal groups and treatment protocols are provided in Table 2. Tumor size and body weight were measured during this period.The tumor volume was measured with calipers and was calculated as V = 0.5 × length × (width) 2 .All mice were sacrificed on the 21st day after tumor implantation, and tumor tissues were collected, weighed, and imaged.The TGI% was calculated using the following formula.
Tumors and major organs, including the heart, liver, spleen, stomach, and kidneys, were collected for histopathological examination through H&E staining.The tumor inhibition rate was calculated according to the average tumor weight (W).TUNEL and Ki67 staining was performed in the selected organs to assess efficiency.Following the TUNEL detection kit's (G1506, Servicebio, Wuhan, China) instructions, the tissue sections were treated with a detection mixture (TDT enzyme, dUTP, and buffer mixed in a 1:5:50 ratio) to perform the labeling.The sections were incubated for 1 h at 37 • C, and then washed 4 times with PBS (pH = 7.4) for 5 min each time.Then, the sections were added to 100 µL of click reaction solution, incubated for 30 min at room temperature, and washed with PBS twice for 5 min each time.Sections were counterstained with Hoechst 33258 for 8 min at room temperature and finally observed under a fluorescence microscope.For Ki-67 immunochemistry, the sections were placed in 0.01 M citrate buffer (pH 6.0) at 100 • C for 10 min for antigen repair.After blocking in 5% sheep serum, sections were incubated with rabbit monoclonal antibody Ki-67 (GB151499, Servicebio, China) at a dilution of 1:500 overnight at 4 • C. Subsequently, the goat anti-rabbit lgG (H+L) secondary antibody conjugated with HRP (1:200; GB23303, Servicebio, China) was applied at room temperature for 40 min.Sections were stained with DAB, then counterstained with hematoxylin, dehydrated, and fixed.

Statistical Analysis
The presented results are representative of three or more independent experiments, with data expressed as means ± standard deviation (SD).All statistical analyses were conducted using GraphPad Prism 7.0.Comparisons between two groups were made using t-tests, while comparisons involving more than two groups used two-way ANOVAs.A p-value < 0.05 was considered statistically significant.

Conclusions
In conclusion, this study highlights the effects of enhancing antibody endocytosis.Both the scFv and the LP are small-protein molecules; the LP, an oligopeptide carrier, effectively traverses cell membranes to deliver substances, while the scFv exhibits antitumor activity.We hypothesized that a fusion molecule combining an LP and scFv could yield improved characteristics.To test this, a novel antibody-based drug, LP-scFv, was engineered by fusing an anti-HER2 scFv with an LP.This fusion enhances membrane penetration and protein transport, thereby increasing the effective internalization and subsequent antitumor activity of the scFv.
The principal findings of this study are as follows: Firstly, the LP-scFv conjugate, successfully expressed in the E. coli system, significantly enhanced the internalization efficiency of the scFv (approximately twofold) while preserving its cell specificity, affinity for the HER2 antigen, and anticancer activity.LP-scFv augmented the inhibition of phosphorylation in the MAPK-ERK1/2 and PI3K/AKT signaling pathways, demonstrating greater suppression of cell proliferation and promotion of apoptosis in vitro.Secondly, in vivo experiments using a xenotransplantation model confirmed that LP conjugation significantly improved scFv's antitumor activity, with LP-scFv's effects comparable to trastuzumab.H&E staining and serum biochemical analyses indicated the low toxicity and good tolerability of both the scFv and LP-scFv.Finally, this study acknowledges certain limitations, such as the need to explore the efficacy of whole antibody conjugated with LP, the effects of LP-scFv in different animal species, and the underlying endocytosis mechanisms of LP-scFv.Overall, our research demonstrates that constructing LP-scFv is an effective strategy to enhance the scFv's antitumor activity both in vivo and in vitro.As an HER2-positive breast cancer-specific drug or a small-molecule drug carrier model, LP-scFv, with its simple process, holds significant potential for application.These findings offer a novel approach to improving internalization efficiency and serve as a reference for designing new, small antibody drugs.

Figure 1 .
Figure 1.The internalization of scFv−EGFP and LP−scFv−EGFP in HER2−positive BT474 cells and HER2−negative MCF−7 cells.Confocal images of internalization after scFv−EGFP and LP−scFv−EGFP binding to BT474 cells (a) and MCF−7 cells (b) with Hoechst 33342 used for nuclear contrast (600×; scale bar, 20 μm).(c) The binding and endocytosis ability of scFv−EGFP or LP−scFv−EGFP to BT474 cells via flow cytometry.Black line: control; blue line: 4 °C; red line: 37 °C.(d) The internalization efficacy of scFv−EGFP or LP−scFv−EGFP in BT474 cells.*** p < 0.001, vs. medium; ### p < 0.001, vs. scFv−EGFP.To confirm the endocytosis of scFv−EGFP and LP−scFv−EGFP into BT474 cells, threedimensional (3D) models were created.BT474 cells, treated with scFv−EGFP or LP−scFv−EGFP and stained with Hoechst 33342 and far-infrared DiD, were analyzed using confocal microscopy.Both scFv−EGFP (Figure 2a(i)) and LP−scFv−EGFP (Figure 2b(i)) were observed near the nucleus (blue), with LP−scFv−EGFP showing a more pronounced distribution.Three-dimensional sections of the BT474 cells showed that a part of scFv−EGFP or LP−scFv−EGFP (green; cyan arrow) was embedded in the cell membrane (red), indicating that this part of the protein bound to the HER2 antigen on the cell membrane and had not been endocytosed into the cell.The other part of the scFv−EGFP or LP−scFv−EGFP protein (green; yellow arrow) was apart from the cell membrane (red) and had access to the nucleus (blue), which indicated that this part of the scFv−EGFP or LP−scFv−EGFP protein had entered the cell (Figure 2a(ii),b(ii)).More importantly, most LP−scFv−EGFP (green; yellow arrow) was between the cell membrane (red) and the nucleus (blue), which indicated that most of the LP−scFv−EGFP protein was endocytosed into the cell.Quantitative fluorescence analysis of BT474 cells (Figure 2a(iii),b(iii)) showed 44.2% overlap of scFv−EGFP with the cell membrane (red), and 21.5% for LP−scFv−EGFP, indicating internalization of these proteins, particularly LP−scFv−EGFP.Laser confocal microscopy results suggest that coupling an LP with the scFv maintained specificity and improved scFv internalization.

Figure 2 .
Figure 2. The confocal 3D section of BT474 cells treated with scFv−EGFP (a) and LP−scFv−EGFP (b).Example Z−projection image and 3D section of the side view of a BT474 cell stained for nucleus, cell membrane, scFv−EGFP (a(i,ii)) and LP−scFv−EGFP (b(i,ii)).Quantitative fluorescence analysis of the cell membrane, nucleus, scFv−EGFP (a(iii)), and LP−scFv−EGFP (b(iii)) along the front-to-back axis (white arrow) of BT474 cells.

Figure 2 .
Figure 2. The confocal 3D section of BT474 cells treated with scFv-EGFP (a) and LP-scFv-EGFP (b).Example Z-projection image and 3D section of the side view of a BT474 cell stained for nucleus, cell membrane, scFv-EGFP (a(i,ii)) and LP-scFv-EGFP (b(i,ii)).Quantitative fluorescence analysis of the cell membrane, nucleus, scFv-EGFP (a(iii)), and LP-scFv-EGFP (b(iii)) along the front-to-back axis (white arrow) of BT474 cells.
. Considering that LP induction promoted scFv internalization and antitumor activity in HER2−positive cells in vitro, the mechanisms were further analyzed through the MAPK−ERK1/2 and PI3K/AKT pathways.The expression levels of apoptosis−related proteins, such as Caspase−3, cleaved Caspase−3, Bax, and Bcl2, were also investigated via Western blotting.The results indicated that BT474 cells treated with the scFv and LP−scFv exhibited significantly increased expressions of Bax and cleaved Caspase−3 and a significant decrease in Bcl2 levels compared to the control group.Notably, these effects were more pronounced with LP−scFv than with the scFv alone (Figure 5a−f).BT474 cells treated with the scFv and LP−scFv showed significant reductions in phosphorylated ERK1/2 (p−ERK1/2) and AKT (p−AKT) levels, with LP−scFv having a stronger effect than the scFv.In line with the flow cytometry results, neither the scFv nor LP−scFv induced apoptotic death or significant changes in AKT or ERK phosphorylation in MCF−7 cells (Figure5g).These findings indicate that LP−scFv enhances the cytotoxic activity and apoptosis of the scFv in BT474 cells through the MAPK−ERK1/2 and PI3K/AKT pathways.Collectively, LP introduction promotes scFv internalization without altering its mechanism of action, improving the scFv's anticancer activity in HER2−positive BT474 cells by increasing its intracellular dosage.
shows no significant changes in liver function indicators (alanine aminotransferase [ALT], aspartate aminotransferase [AST], total proteins [TP], albumin [ALB]) or renal function biomarkers (creatinine [CRE], blood urea [UREA], blood uric acid [UA]) in these treatment groups.These results suggest that the scFv and LP-scFv demonstrated effective therapeutic efficacy without significant toxicity to major organs in mice.andeosin (H&E) staining.Additionally, serum samples from the control and experimental groups were analyzed for liver and kidney function tests.Figure7aindicates no significant morphological differences in the trastuzumab, scFv, and LP−scFv groups compared to the control group.Similarly, Figure7bshows no significant changes in liver function indicators (alanine aminotransferase [ALT], aspartate aminotransferase [AST], total proteins [TP], albumin [ALB]) or renal function biomarkers (creatinine [CRE], blood urea [UREA], blood uric acid [UA]) in these treatment groups.These results suggest that the scFv and LP−scFv demonstrated effective therapeutic efficacy without significant toxicity to major organs in mice.

Figure 7 .
Figure 7.The systemic toxicity of trastuzumab, scFv, and LP−scFv in tumor-bearing mice.(a) H&E staining images (200×) for major tissues (heart, liver, spleen, stomach, and kidney) from the mice treated in the control group (PBS) and treatment groups (trastuzumab, scFv, and LP−scFv) (Scale bar, 100 μm) and the identification characteristics were pointed with an arrow.(b) Liver function indices (ALT, AST, TP, and ALB levels) and kidney function indices (CRE, UA, and UREA levels).Data are represented as the means ± SD; ns, no significance.

Figure 7 .
Figure 7.The systemic toxicity of trastuzumab, scFv, and LP-scFv in tumor-bearing mice.(a) H&E staining images (200×) for major tissues (heart, liver, spleen, stomach, and kidney) from the mice treated in the control group (PBS) and treatment groups (trastuzumab, scFv, and LP-scFv) (Scale bar, 100 µm) and the identification characteristics were pointed with an arrow.(b) Liver function indices (ALT, AST, TP, and ALB levels) and kidney function indices (CRE, UA, and UREA levels).Data are represented as the means ± SD; ns, no significance.

Table 1 .
Fitted binding parameters, on-rate (k on ), off-rate (k off ), and K D for scFv and LP-scFv.

Table 2 .
The animal groups and treatment protocols.