Fusion of apoptosis‐related protein Cytochrome c with anti‐HER‐2 single‐chain antibody targets the suppression of HER‐2+ breast cancer

Abstract Cancer treatment has gradually developed from toxic chemotherapy to targeted therapy with fewer side effects. Approximately 30% of breast cancer patients overexpress human epidermal growth factor receptor 2 (HER‐2). Previous studies have successfully produced single‐chain antibodies (scFv) targeting HER‐2+ breast cancer; however, scFv have poor stability, easy aggregation and a shorter half‐life, which have no significant effect on targeting therapy. Moreover, scFv has been considered as a drug delivery platform that can kill target cells by effector molecules. However, the functional killing domains of immunotoxins are mainly derived from plant or bacterial toxins, which have a large molecular weight, low tissue permeability and severe side effects. To address these concerns, we designed several apoptotic immune molecules to replace exogenous toxins using endogenous apoptosis‐related protein DNA fragmentation factor 40 (DFF40) and tandem‐repeat Cytochrome c base on caspase‐3 responsive peptide (DEVD). Our results suggest that DFF40 or Cytc fusion scFv specifically targets HER‐2 overexpressing breast cancer cells (SK‐BR‐3 and BT‐474) rather than HER‐2 negative cells (MDA‐MB‐231 and MCF‐7). Following cellular internalization, apoptosis‐related proteins inhibited tumour activity by initiating endogenous apoptosis pathways, which significantly reduced immunogenicity and toxic side effects. Therefore, we suggest that immunoapoptotic molecules may become potential drugs for targeted immunotherapy of breast cancer.

overexpressing patients also have correspondingly lower survival rates and faster relapse. [6][7][8] Recently, antibody-based immunotherapy for cancer has grown in popularity due to its specific targeting and remarkable efficacy. Clinically, the humanized monoclonal antibody, trastuzumab, has primarily been used to treat HER-2 overexpressing metastatic breast cancer. Trastuzumab attaches itself to HER-2 to block ligand binding and down-regulate both angiogenic and metastatic genes. [9][10][11] Despite the significant efficacy of trastuzumab, several patients continue to develop resistance and disease progression. [12][13][14][15] Single-chain antibodies (scFv) are small molecule fragments formed by linking the heavy-chain variable region (VH) and light-chain variable region (VL) with a short (15 aa-20 aa), flexible peptide that has excellent targeting and reduced immunogenicity. The recombinant immunotoxin constructed with scFv as a vector-induced cell death through the inhibition of toxin-mediated translation; however, various toxins (e.g. pseudomonas exotoxin, staphylococcal enterotoxin and ribosomeinactivating protein gelonin) have strong side effects, including gastrointestinal toxicity, liver toxicity, hypoalbuminemia and vascular leakage syndrome. [16][17][18] To reduce these side effects, we incorporated endogenous apoptosis-related proteins instead of plant or bacterial toxins with scFv, thereby inhibiting tumour progression by initiating apoptosis.
Apoptosis is an essential means by which the homeostasis of multicellular organisms is maintained and can selectively eliminate potentially harmful or infected cells. [19][20][21] The accepted biological markers of apoptosis include chromatin concentration and DNA fragmentation, 22,23 which is primarily triggered by activation of the DNA fragmentation factor, DFF40. DFF40, known as caspase-activated DNase (CAD), has been identified as a downstream molecule of caspase-3. In addition, DFF40 and its natural inhibitor molecule, DFF45, typically constitute heterodimers. Previous studies indicate that upon DFF45 cleavage by activated caspase-3, DFF40 was isolated and inhibition of its nuclease activity was lifted, which subsequently degrades double-stranded DNA. 24 DFF40 fusion expression and gonadotropin-releasing hormone (GnRH) have been used as targeted therapy for adenocarcinoma. 25 In addition, Cytochrome c (Cytc) also represents a key molecule involved in apoptosis. 26 In the mitochondrial-induced endogenous apoptotic pathway, after cells respond to apoptotic stimuli, the mitochondrial outer membrane is permeabilized, and Cytc is released into the cytoplasm to bind to apoptotic enzyme activating factor 1 (Apaf-1). After recruiting pro-caspase-9, they form a giant Dalton complex: apoptotic bodies, which can promote caspase-3 activation and induce apoptosis as described above. 27,28 Unlike necrosis, 29 cells shrink following apoptosis, without the swelling and rupturing of organelles and plasma membranes. The cell membrane structure remains intact, and apoptotic cells eventually split into multiple apoptotic bodies wrapped in membranes, which can be quickly phagocytosed and cleared by phagocytes residing nearby. 30 Such a process is not associated with many adverse effects (e.g. inflammatory reactions) and is a safer means of inhibiting tumour progression. This study aimed to construct immunoapoptotic molecules by fusing anti-HER-2 scFv as a binding domain with DFF40 or tandem-repeat Cytc (3Cytc) and evaluate the anti-tumour efficacy of these constructs. The human embryonic kidney cell line, HEK-293T (ATCC®CRL-11268),   and human breast cancer cell lines, SK-BR-3 (ATCC®HTB-30 All cells were cultured in DMEM medium (Gibco) supplemented with 10% FBS (Gibco) and a 1% mixture of penicillin and streptomycin (Invitrogen). The cells were routinely tested for mycoplasma contamination with negative results. All cells were grown at 37°C in a humidified atmosphere containing 5% CO 2 .

| Construction of recombinant expression plasmids
The gene encoding anti-HER-2 scFv was composed of the heavychain variable region (VH) and the light-chain variable region (VL) of trastuzumab, which were connected by a flexible peptide linker, 3 . The scFv cDNA sequence was artificially synthesized and cloned into the prokaryotic expression vector, pET-32a(+), thereby creating the recombinant expression plasmid termed pET-32a(+)-scFv. Either a DFF40 or Cytc coding sequence was inserted upstream of scFv through Bgl II and Nco I to construct the expression plasmid of the fusion protein, DFF40-scFv or Cytc-scFv. 3Cytc-scFv was designed by simultaneously connecting three Cytc molecules through the caspase-3 cleavage site, DEVD, and cloned into pET-32a(+)-scFv as described above. A list of the primers used for homologous recombination are listed in Table 1

| Immunofluorescence
Cells were incubated with 5 µg/ml of various protein constructs.
After the non-specific binding sites were blocked with 4% BSA, the cells were incubated with mouse anti-6His mAb (1:100 dilution) followed by an incubation with a goat anti-mouse IgG-FITC  According to the instructions, CCK-8 (Dojindo) reagent was added, incubated and the absorbance was immediately measured at 450 nm using a microplate reader (Bio-Teck). Each experimental group was performed in triplicate.

| Crystal violet staining
Cells were subsequently fixed with 4% paraformaldehyde and reacted with a 1 ml crystal violet staining solution (Solarbio). Cell proliferation activity was evaluated by comparing the number of stained cells.
2.5 | Apoptosis assays: morphological observations, apoptosis-related protein analysis and flow cytometry 2.5.1 | Caspase-3 activity assay Cells stimulated as described above were digested and lysed.
According to the manufacturer's instructions, the reaction solution and caspase-3 cutting substrate-Ac-DEVD-pNA (Jcbio) were added to establish the reaction system. The absorbance at 405 nm was measured using a microplate reader. Each experimental group was performed in triplicate.

| Level of Bcl-2 and Bax expression
Cells treated with 100 nM of the different protein constructs were lysed with RIPA and the total protein concentration was confirmed using a BCA detection kit (Thermo). The denatured proteins were transferred to PVDF membranes as described above and incubated with antibodies specific to Bcl-2 and Bax (CST) (1:1000 dilution). αtubulin (CST) (1:1000 dilution) was used as the internal reference.

| Annexin V-FITC/PI double staining
A total of 5 × 10 5 cells per well were seeded into six-well plates and treated as described above. The cells were digested with EDTA-free trypsin, collected and resuspended binding solution. Annexin V-FITC and PI were added in sequence and incubated together with the cells in the dark. The processed samples were immediately detected using a BD FACS Calibur (BD Biosciences). The results were analysed with FlowJo software.

| Morphological changes
SK-BR-3 cells were seeded onto 9.6 cm 2 coverslips overnight to achieve approximately 60% confluency and stimulated as

| Statistical analysis
Data analysis was conducted using the statistical program SPSS v.20.0 (IBM). All results were expressed as the mean ± SD. The statistical differences between groups were analysed by Student's t test and anova. A p value of <0.05 was considered significant.

| Preparation and identification of different protein constructs
Artificially synthesized cDNA fragments were cloned into pET-32a(+), a vector expressing soluble proteins via TrxAtag, to construct recombinant plasmids. The target genes consisted of scFv alone and three fusion constructs: DFF40-scFv, Cytc-scFv and 3Cytc-scFv. All plasmid maps are shown in Figure 1A-D, with different colours representing different structural units. Figure 1E shows a two-dimensional structure diagram clearly displayed for each component. All constructed recombinant plasmids were sequenced and demonstrated to harbour the correct coding sequence.
Target gene expression was controlled by the bacteriophage T7 promoter and lactose operon. The optimal induction conditions of E. coli were determined by the control variable method as 0.5 mM IPTG and a low temperature reaction for 12 h at 20°C.
SDS-PAGE confirmed that the purified proteins eluted from the Ni-NTA column were primarily in the 100 mM imidazole eluent ( Figure 1F). Based on the optical density analysis of the band, the purity of each protein was greater than 90%. After removing imidazole by dialysis, each protein construct was concentrated to 1-2 mg/ml in an ultrafiltration tube. Western blotting was performed to confirm protein expression via an anti-6His antibody, and the specific combination was clearly visible at the corresponding position on the PVDF membrane. All recombinant plasmids successfully expressed target proteins following transformation into E. coli BL21 (DE3), with correct molecular weights of 46 kDa for scFv; 84 kDa for DFF40-scFv; 58 kDa for Cytc-scFv and 83 kDa for 3Cytc-scFv ( Figure 1G).

| scFvs bind specifically to HER-2+ breast cancer cells
The targeting activity of scFv was investigated with an immunofluorescence analysis. Since breast cancer cells are divided into differ-

| Selective cytotoxicity of immunoapoptotic molecules to different tumour cells
Next, we closely monitored the viability of cultured cells exposed to each of the four respective constructs for 48 h. As shown in Figure  Therefore, the presence of scFv was associated with the specific toxicity of immunoapoptotic molecules towards cells overexpressing HER-2 without affecting the activity of normal cells.

| DFF40 and Cytc induce target cell apoptosis
Previous studies have suggested that caspase-3 is an executor of the caspase family and plays a key role in the apoptosis pathway. 27,31,32 Here, the caspase-3 specific cleavage sequence, DEVD, was coupled to the yellow group p-nitroaniline, was released when the substrate was cleaved by activated caspase-3, and could be detected at 405 nm. We measured the level of caspase-3 activity to indirectly investigate whether scFv-delivered DFF40 and Cytc could induce the apoptosis of target cells. As shown in Bax. If the level of Bax expression is higher than that of Bcl-2, it will promote apoptosis, otherwise apoptosis will be inhibited. 35,36 In SK-BR-3 cells, the level of Bcl-2 in the cytoplasm was significantly reduced under the stimulation of DFF40-scFv, and Bax was simultaneously increased. Moreover, the Bcl-2/Bax ratio was confirmed to be reduced by quantification ( Figure 3E). Similar to the trends observed in previous experiments, the Bcl-2/Bax ratio under 3Cytc-scFv stimulation also decreased, but to a lesser extent compared with that of DFF40-scFv, whereas Cytc-scFv did not have a substantial effect. Together, these data indicate that single scFv could not induce apoptosis, and only exerted anti-tumour activity when it was coupled with DFF40 or Cytc.

| Significant morphological features of apoptosis
Apoptosis is associated with obvious morphological characteristics, including a reduced cell size, nuclear shrinkage, membrane bubbles, chromatin condensation, cytoskeletal disintegration and the formation of apoptotic bodies. 37,38 Although there are many methods that can be used to detect apoptosis, morphological observations are still reliable. SK-BR-3 cells stimulated with different protein constructs were stained with Hoechst to label DNA and observed with a fluorescence microscope. As shown in Figure 5A, the cells under the influence of single scFv exhibited normal nuclei. In contrast, DFF40-scFv was associated with ruptured nuclei and a colour change from blue to bright white, which represented the typical nuclear characteristics given that the chromatin shrunk during apoptosis. Consistent with the above findings, a similar phenomenon was observed in the 3Cytc-scFv-treated cells, whereas the nuclear changes in the Cytc-scFv-treated cells were slightly visible.

| Immunoapoptotic molecules inhibit breast tumours in vivo
To confirm the clinical relevance of the suppression induced by the constructed immunoapoptotic molecules in HER-2+ breast cancer, we subcutaneously injected SK-BR-3 cells into BALB/c nude mice to establish a breast cancer xenograft model. Proteins were administered intraperitoneally to treat the tumour-bearing mice ( Figure 6A).
Tumour progression in mice treated with fusion proteins, DFF40-Cytc, 3Cytc-scFv and Cytc-scFv, was significantly suppressed compared with that of the scFv control group, and the anti-tumour effects of the three were diminishing ( Figure 6B-D). Images of TUNEL-stained cells also showed different degrees of apoptosis in the tumour tissue between groups ( Figure 6E), of which the DFF40-scfv group exhibited the largest proportion of apoptotic cells. These data were consistent with the results of the in vitro experiments, indicating that the immunoapoptotic molecules DFF40-scFv, Cytc-scFv and 3Cytc-scFv showed targeted suppression of HER-2+ breast cancer cells in vivo. Importantly, DFF40 was associated with more potent antitumour activity compared to that of Cytc, and the introduction of caspase-3 cleavage site to tandem-repeat Cytc enhanced such activity.

| DISCUSS ION
Cancer immunotherapy has attracted increased attention in recent years. Moreover, the combination of toxins and recombinant antibodies immunogenicity induced by immunotoxins can be avoided. In addition, traditional anti-HER-2 therapeutic mAbs rely on the patient's immune system or block the HER-2 signaling pathway to exert an tumour killing effect. 47 Moreover, at least 70% of patients with HER-2 positive breast cancer exhibit disease progression. 48 Therefore, immunoapoptotic molecules can bypass the endogenous resistance mechanism, thereby greatly reducing the resistance to mAb-based drugs.
In addition to DFF40-scFv and Cytc-scFv, we creatively constructed tandem-repeat Cytochrome c (3Cytc-scFv). It is widely known that the final step in the apoptosis cascade is the activation of the caspase-3 executive protein, which inhibits DNA repair and initiates DNA degradation. 49,50 Moreover, caspase-3 is known as a cysteine-containing aspartate proteolytic enzyme, which specifically recognizes and cleaves the peptide bond on the aspartic acid residue of the target protein. Our results showed that the cleavage sequence of caspase-3 was Asp-Glu-Val-Asp (DEVD), 51,52 and 3Cytc-scfv was formed by connecting three Cytc proteins in a series through DEVD. The results showed that fusion proteins can specifically bind to breast cancer cells overexpressing HER-2 in vitro. Under the same treatment conditions, DFF40-scFv was the most toxic, resulting in the greatest degree of apoptosis, whereas only a weak effect was observed for Cytc-scFv. It was gratifying that even though the activity of 3Cytc-scfv with the introduced caspase-3 cleavage site was inferior to that of DFF40-scFv, it was significantly better than Cytc-scFv and could induce substantial apoptosis. Similar findings were obtained in vivo.
As a downstream molecule of the apoptosis cascade, the activation of DFF40 will lead to irreversible DNA damage, thereby quickly killing the cells. 24 However, Cytc triggers a response process, which is first released from the mitochondria into the cytoplasm to form an apoptosome, which then activates apoptotic hydrolases. [26][27][28] We considered that although DFF40-scFv has a stronger tumour killing rate, it was also the most toxic and has a molecular weight that is too large to have a negative impact on the body, making it a less than ideal construct. Moreover, Cytc has a smaller molecular weight, fewer side effects, and displayed better pro-apoptotic efficacy than DFF40. When 3Cytc-scFv is internalized into the target cell and the fusion protein induces apoptosis, activated caspase-3 can recognize the DEVD amino acid sequence between Cytc and cut it off. As the DEVD breaks, each 3Cytc-scFv construct will simultaneously release three free Cytc molecules. This mechanism is thus a clever virtuous cycle that activates the apoptosis pathway and in turn perpetuates the effect. Importantly, 3Cytc-scFv can only limit its anti-tumour effects to apoptosis, thereby avoiding toxicity and other side effects, which are not available for DFF40-scfv and many other types of immunoapoptotic molecules.
We can safely conclude that the single-chain antibody recombinant immune molecules fused with apoptotic proteins constructed in this study effectively targeted the breast cancer surface receptor, HER-2, and induced apoptosis both in vivo and in vitro. No toxic effects were detected in the HER-2 negative cells. To the best of our knowledge, there are few reports investigating the combination of the apoptotic protein, DFF40, and a single-chain antibody for the treatment of HER-2 positive breast cancer. Importantly, 3Cytc-scFv introduced with caspase-3 restriction sites represents an important supplement to the strategy of treating tumours through endogenous apoptosis pathways. As an ideal immunoapoptotic molecule in this study, although 3Cytc-scFv provides a more novel and effective tumour antibody treatment approach, outstanding problems remain to be solved prior to clinical application. First, we can improve the purification process to eliminate possible contaminants. Secondly, it is necessary to further optimize the dosing regimen in rodents and try to implement recombinant antibodies for the treatment of nonhuman primates. Finally, according to the design concept for 3Cytc-scFv, new small endogenous effector proteins can be developed for the combined use with Cytc. The above optimization and mechanistic research may lead to more effective anti-tumour activity.
In summary, we combined single-chain antibodies with apoptosis and prepared a variety of immunoapoptotic molecules by cloning technology. The 3Cytc-scFv strategy of connecting multiple small pro-apoptotic molecules in a series and releasing them through enzymatic cleavage demonstrated superior tumourspecific killing activity with fewer side effects, which is in line with the concept of drug treatments with a high efficiency and low toxicity. Therefore, these findings support targeting tumour antibody pathways to induce apoptosis in the future of cancer treatment.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data used to support the findings of this study are available from the corresponding authors upon request.