Construction of a versatile fusion protein for targeted therapy and immunotherapy

Abstract

Antibody (Ab)‐based drugs have been widely used in targeted therapies and immunotherapies, leading to significant improvements in tumor therapy. However, the failure of Ab therapy due to the loss of target antigens or Ab modifications that affect its function limits its application. In this study, we expanded the application of antibodies (Abs) by constructing a fusion protein as a versatile tool for Ab‐based target cell detection, delivery, and therapy. We first constructed a SpaC Catcher (SpaCC for short) fusion protein that included the C domains of Staphylococcal protein A (SpaC) and the SpyCatcher. SpaCC conjugated with SpyTag‐X (S‐X) to form the SpaCC‐S‐X complex, which binds non‐covalently to an Ab to form the Ab‐SpaCC‐S‐X protein complex. The “X” can be a variety of small molecules such as fluoresceins, cell‐penetrating peptide TAT, Monomethyl auristatin E (MMAE), and DNA. We found that Ab‐SpaCC‐S‐FITC(−TAT) could be used for target cell detection and delivery. Besides, we synthesized the Ab‐SpaCC‐SN3‐MMAE complex by linking Ab with MMAE by SpaCC, which improved the cytotoxicity of small molecule toxins. Moreover, we constructed an Ab‐DNA complex by conjugating SpaCC with the aptamer (Ap) and found that Ab‐SpaCC‐SN3‐Ap boosted the tumor‐killing function of T‐cells by retargeting tumor cells. Thus, we developed a multifunctional tool that could be used for targeted therapies and immunotherapies, providing a cheap and convenient novel drug development strategy.

1. INTRODUCTION

Antibody drugs are a large class of biological therapies, and over 100 Ab‐based therapeutics have been approved for the treatment of human diseases because of their stability, specificity, and suitability for protein engineering (Heo, 2022; Lyu et al., ²⁰²²). Most approved Abs are in IgG format, although several alternatives are emerging, including bispecific antibodies (BsAbs), IgG mixtures, and Ab fusion proteins (Shin et al., 2021). However, the applications of Ab‐based drugs are limited. Therefore, the development of tools for Ab‐based detection, delivery, and therapy is important for Ab therapeutics.

Staphylococcal protein A (Spa) is a surface protein present in staphylococci that contains five homologous domains, named E, D, A, B, and C, from the N to C termini (Deis et al., 2014; Rigi et al., ²⁰¹⁹). Spa can specifically bind to the Fc segment of IgG molecules in mammals (except for IgG3), allowing the Fab segment to specifically bind to antigens (Jansson et al., 1998; Lee et al., ²⁰²¹). The E, A, B, and C domains of Spa, as useful tools, have been widely used in medical and biotechnological fields, such as immunoprecipitation (IP), enzyme‐linked immunosorbent assay (ELISA), and western blotting (WB) (Guillon et al., 2018; Schmidt et al., ²⁰²²). Ab‐based target cell detection, delivery, and therapy require binding or labeling with Abs, and the C domain of Spa (SpaC) is an ideal protein because it is thought to be the most stable of the domains (Minakuchi et al., 2013).

The SpyCatcher‐SpyTag system is a recently developed protein ligation method. SpyCatcher, which recognizes homologous 13‐amino acid peptides (SpyTag), is a modified domain derived from Streptococcus pyogenes surface protein of Streptococcus pyogenes (Reddington & Howarth, 2015). SpyCatcher and SpyTag form a covalent copeptide bond between the lysine in SpyCatcher and the aspartate side chains in SpyTag. The SpyCatcher‐SpyTag system has been used to create covalently stable multiprotein complexes for modular vaccine production and to label proteins (e.g., for microscopy) (Hatlem et al., 2019; Pessino et al., ²⁰¹⁷; Wang et al., ²⁰¹⁹). This system relies on a high stability that is not reversed by the addition of other peptides and does not affect the initial catalytic activantiity and function of the target protein (Du et al., 2023; Sun et al., ²⁰¹⁹). Furthermore, SpyCatcher can be fused to reporter proteins such as GFP (Song et al., 2022). SpyTag is versatile because it is a short, unfolded peptide that can be fused to the exposed location of the target protein. Moreover, SpyTag can be easily synthesized and modified by chemical groups such as Azide (‐N3) and Fluorescein (FITC) (Alam et al., 2019; Wang et al., ²⁰²²; Zakeri et al., ²⁰¹²). However, the application of the SpyCatcher‐SpyTag system coupled with Ab‐based drugs in targeted immunotherapies remains unclear.

In this study, we constructed a versatile fusion protein (SpaC Catcher fusion protein, SpaCC) containing the SpyCatcher domain (conjugated with the versatile SpyTag) and the C domains of Staphylococcal protein A (Ab‐binding domain) (Scheme 1). Ab was bound with SpaCC‐S‐FITC(‐TAT) to form an Ab complex for targeted cell detection and delivery. Furthermore, Ab was bonded with SpaCC‐SN3‐MMAE(‐Aptamer) to yield an Ab‐drug/DNA complex for targeted therapy and immunotherapy. Thus, we developed a multifunctional tool that can be used for targeted therapies and immunotherapies, providing a cheap and convenient new drug development strategy.

SCHEMA 1.

SpaCC conjugated with SpyTag‐X (S‐X) to form the SpaCC‐S‐X complex, which binds non‐covalently to an Ab Fc fragment to form the Ab‐SpaCC‐S‐X protein complex, which is used in cell detection, cell delivery, target therapy and immunotherapy. The “X” can be a variety of small molecules such as FITC, TAT, MMAE, and DNA.

2. RESULTS AND DISCUSSION

2.1. Construction of Ab‐SpaCC‐S‐X protein complex

Spa is safe in crab‐eating monkeys (Bernton & Haughey, 2014) and has been effectively utilized in the treatment of idiopathic thrombocytopenic purpura (ITP) disease (Kapur et al., 2018). Thus, we chose the C domain of Spa (SpaC) as a basic structural unit to establish a versatile protein complex and constructed the sequence of a fusion protein, named SpaC Catcher or SpaCC for short, which consisted of a SpyCatcher domain labeled with 6‐His, a protein linker, and the C domain of Staphylococcal protein A (Figure 1a). The protein sequences are listed in Table S1. We then inserted the sequence into the pET28a plasmid and expressed SpaCC in BL21(DE3) E. coli competent cells. SpaCC was purified by nickel magnetic beads binding with His‐tagged and identified by Coomassie blue staining. As shown in Figure 1b, the SpaCC was successfully constructed and purified. To verify whether SpaCC could bind to Abs, we coated 1 μg rituximab (anti‐CD20 Ab) or trastuzumab (anti‐HER2 Ab) in a 96‐well plate for ELISA detection. SpaCC proteins were added at mole proportions to Ab of 1:0.01, 1:0.1, 1:1, and 1:10, individually. The results showed that the binding effect was gradually enhanced with the increase of SpaCC protein concentration, and when Ab: SpaCC for 1:1, Ab and SpaCC could reach complete binding (Figure 1c). To further construct a functional protein complex, we conjugated the SpaCC protein with SpyTag‐X to synthesize a new protein complex, which was named SpaCC‐S‐X, for example, SpaCC‐S‐FITC (Figure 1d). Finally, we connected the SpaCC‐S‐X complex and Abs to form an Ab‐protein complex (Ab‐SpaCC‐S‐X) and tested its various applications in subsequent experiments (Scheme 1).

FIGURE 1.

Construction of SpaCC and Ab‐SpaCC‐S‐X protein complex. (a) Vector construction of SpaCC: SpaC, linker, SpyCatcher, and 6‐His. (b) Purified SpaCC was identified by Coomassie blue staining. (c) Antibodies (Rituximab or Trastuzumab) binding to SpaCC were determined by ELISA. Ctrl: Rituximab or Trastuzumab coated in 96‐well plates without SpaCC (d) The SpaCC‐S‐FITC‐coupled protein was verified using Coomassie blue staining. (e) Schematic representation of the Ab‐SpaCC‐S‐FITC protein structure, including CD20‐SpaCC‐S‐FITC and HER2‐SpaCC‐S‐FITC. (f) Flow cytometry analysis of CD20 expression in Nalm6, Jurkat, Ramos, and SU‐DHL‐4 cells, and HER2 expression in HEK293T, MDA‐MB‐231, and SK‐BR‐3 cells; blank: without staining. (g) The binding capacity of CD20/HER2‐SpaCC‐S‐FITC to tumor cells was detected by flow cytometric assay; Ctrl: SpaCC‐S‐FITC.

2.2. Ab‐SpaCC‐S‐FITC enables the detection of target cells

Tumor cell‐surface antigens are usually detected using fluorescently labeled antibodies (Rodig, 2021). However, commercial therapeutic Abs cannot be used to test the antigens of target cells because of the lack of fluorescent labeling. To display the feasibility of the coupling system more visually, we used in‐gel fluorescence photography to compare the sizes of SpaCC and SpaCC‐S‐FITC bands and found that fluorescence (FITC) had been successfully conjugated with SpaCC (Figure S1A–C), indicating that the initial activity and function of the protein were not affected after coupling. To determine whether the Ab‐SpaCC‐S‐X complex could be used to detect antigens and recognize target cells specifically, we mixed the SpaCC‐S‐FITC complex with rituximab or trastuzumab to form Ab‐SpaCC‐S‐FITC protein complexes, such as CD20‐SpaCC‐S‐FITC and HER2‐SpaCC‐S‐FITC (Figure 1e). We first assessed the expression of CD20 or HER2 in tumor cell lines, such as Nalm6, Jurkat, Ramos, SU‐DHL‐4, HEK293T, MDA‐MB‐231, and SK‐BR‐3, and found that CD20 expressed in the human lymphoma cell lines Ramos and SU‐DHL‐4, while HER2 expressed in the human breast cancer cell line SK‐BR‐3 (Figure 1f). We then co‐incubated Ab‐SpaCC‐S‐FITC complex with tumor cells at 37°C for 30 min and demonstrated that the CD20‐SpaCC‐S‐FITC and HER2‐SpaCC‐S‐FITC complex could bind specifically with CD20⁺ and HER2⁺ tumor cells (Figure 1g). Moreover, we compared the labeling effect of standard Abs with Ab‐SpaCC‐S‐FITC (Figure S2A,B), and demonstrated that Ab‐SpaCC‐S‐FITC required a higher concentration to detect the target cells than the standard Abs. Taken together, these findings imply that SpaCC acts as an intermediary connector and that the Ab‐SpaCC‐S‐FITC complex could detect target cells efficiently.

2.3. Ab‐SpaCC‐S‐TAT delivers antibodies into target cells

TAT peptide is a cell‐penetrating peptide derived from the trans‐activating transcriptional activator (TAT) from HIV‐1 and is commonly used as a cell delivery tool that transports DNA, proteins (including peptides and antibodies), and drug molecules across cellular membranes (Koo et al., 2022; Zou et al., ²⁰¹⁷). To efficiently deliver Abs into cells, we linked the SpaCC with the SpyTag‐TAT (containing SpyTag and TAT peptide) to yield the SpaCC‐S‐TAT protein complex, which was identified by Coomassie blue staining with the MW ~35 kD (Figure 2a). We then mixed the Ab (PE anti‐mouse CD45.1 or FTIC anti‐mouse CD45.2 antibody) with SpaCC‐S‐TAT to form the Ab‐SpaCC‐S‐TAT complex at a 1:10 ratio (Figure 2b), which was then added to the tumor cell lines Ramos, Nalm6, and Jurkat. We found that only the co‐incubation of Ab‐SpaCC‐S‐TAT and tumor cell lines at 37°C exhibited fluorescence enhancement at 24 h (Figures S2A,B) and 48 h (Figure 2c,d). Similar results were obtained by confocal microscopy imaging (Figure 2e,f). With time extension, only the Ab‐SpaCC‐S‐TAT group could increase the positive rate, verifying the stability of the protein coupling system. These data suggest that SpaCC acts as an intermediate carrier to connect the Ab and TAT, allowing TAT to successfully deliver the Ab into the cells.

FIGURE 2.

Construction of Ab‐SpaCC‐S‐TAT for delivering antibodies into target cells. (a) The SpaCC‐S‐TAT coupled protein was verified by Coomassie blue staining. (b) Schematic representation depicts the protein structure of Ab‐SpaCC‐S‐TAT, such as CD45.1‐SpaCC‐S‐TAT and CD45.2‐SpaCC‐S‐TAT. (c,d) The Ab‐SpaCC, Ab‐SpyTag‐TAT, and Ab‐SpaCC‐S‐TAT complexes were co‐incubated with Ramos and Jurkat cells for 48 h by flow cytometry analysis. Ab is PE anti‐mouse CD45.1 (c) or FITC anti‐mouse CD45.2 (d). Ctrl: PE anti‐mouse CD45.1 or FITC anti‐mouse CD45.2 antibody. (e,f) Ab‐SpaCC, Ab‐SpyTag‐TAT, and Ab‐SpaCC‐S‐TAT complexes were co‐incubated with Ramos and Jurkat cells for 48 h. Ctrl: PE anti‐mouse CD45.1 antibody (e) or FITC anti‐mouse CD45.2 antibody (f). Blue: Hoechst stained nucleus. Red: PE fluorescent antibody. Green: FITC fluorescent antibody.

2.4. Ab‐SpaCC‐SN3‐MMAE is used for targeted therapy

Next, we investigated whether the SpaCC could be used for targeted therapy by coupling with small‐molecule toxins. MMAE is a small molecule toxin that cannot be used alone due to its severe toxicity, instead, it is often used in Ab‐drug conjugates (ADC) by conjugating with monoclonal antibodies (mAbs) (Chang et al., 2021). For this reason, MMAE is usually purchased with a Dibenzocycloecten (DBCO) modifier, which could react with azide (‐N3) by click chemistry reaction (He, Liu, et al., ^2023b). To conjugate SpaCC with DBCO‐MMAE, we first ligated SpaCC with SpyTag‐N3 to form the SpaCC‐SN3 complex (Figure S4A), and then the complex reacted with DBCO‐MMAE to form the SpaCC‐SN3‐MMAE complex (Figure S4B). We next mixed the Ab (Rituximab or Trastuzumab) with SpaCC‐SN3‐MMAE to form Ab‐drug complexes Ab‐SpaCC‐SN3‐MMAE, which were referred to as CD20‐SpaCC‐SN3‐MMAE and HER2‐SpaCC‐SN3‐MMAE (Figure 3a,b).

FIGURE 3.

Construction of Ab‐SpaCC‐SN3‐MMAE for target therapy. (a) Schematic representation depicts the protein structure of Ab‐SpaCC‐SN3‐MMAE. (b) Schematic diagram of targeted killing of tumor cells by Ab‐SpaCC‐SN3‐MMAE. (c–f) SpaCC‐SN3‐MMAE was mixed with mAbs (Rituximab or Trastuzumab), and the cytotoxicity of Ab‐SpaCC‐SN3‐MMAE was assayed at different concentrations for 48 h (c,d) or 72 h (e,f). (g) HEK293T or SK‐BR‐3 cells were treated with a final concentration of 1 nM in each group for 48 h, and apoptosis was analyzed by flow cytometry. Ctrl: no drug treatment. (h) Nalm6 or Ramos cells were treated with final concentrations of 1, 10, and 100 nM for 48 h in each group, and apoptosis was analyzed by flow cytometry. Ctrl: no drug treatment.

We first evaluated the effect of Ab‐SpaCC‐SN3‐MMAE on the cell cycle of target tumor cells by treating Nalm6 or Ramos cells with 20 nM CD20‐SpaCC‐SN3‐MMAE and HEK293T or SK‐BR‐3 with 1 nM HER2‐SpaCC‐SN3‐MMAE for 48 h, respectively. Flow cytometric analysis showed that the Ab‐SpaCC‐SN3‐MMAE complex led to a significant loss of G1 DNA content and an increase in G2/M DNA content (Figure S3A,B). We then verified the cytotoxicity of Ab‐SpaCC‐SN3‐MMAE on target cells in vitro and found that the Ab‐SpaCC‐SN3‐MMAE complex significantly killed tumor target cells at 48 h (Figures 2d and 3c) and 72 h (Figure 3e,f) in a concentration‐dependent manner. The IC50 values of Ab‐SpaCC‐SN3‐MMAE against CD20⁺ and HER2⁺ tumor cells were 20 nM and 0.5 nM, respectively. Furthermore, the Ab‐SpaCC‐SN3‐MMAE complex induced the cell apoptosis of HER2⁺ SK‐BR‐3 (Figure 3g) and CD20⁺ Ramos (Figure 3h), but not in HER2⁻ HEK293T and CD20⁻ Nalm6 cells at low concentrations. Collectively, these results suggest that Ab‐drug complexes formed by Ab‐binding SpaCC‐SN3‐MMAE can deliver MMAE into tumor cells for targeted therapy, which improves the targeting of small‐molecule toxins, thereby enhancing their anti‐tumor effects.

2.5. Ab‐SpaCC‐SN3‐Ap overcomes anti‐tumor immune escape

Aptamers are single‐stranded oligonucleotides that can bind to targets with high specificity and affinity (Kinghorn et al., 2017). Although the use of BsAbs such as CD19/CD3, CD79b/CD3, CD20/CD3, and BCMA/CD3 has made significant progress in the treatment of relapsed/refractory (R/R) hematological malignancies, the loss of antigen is still a major cause of disease recurrence (Liu et al., 2022; Thakur et al., ²⁰¹⁸). Therefore, we further explored the effect of SpaCC protein to bind antibody to aptamers for immunotherapy. Protein‐tyrosine kinase 7 (PTK7) is highly expressed in various tumor cells and promotes their growth (Chen et al., 2018; Song et al., ²⁰²⁰). We first verified the expression of PTK7 in several tumor cell lines, such as acute T‐lymphoblastic leukemia, acute B‐lymphoblastic leukemia, and Burkitt's lymphoma, by western blotting (Figure 4a) and confirmed the specific binding of the Sgc8 aptamer to Nalm6 or Jurkat target cells by flow cytometry (Figure 4b). To overcome the escape of tumor immunotherapy due to the loss of target antigens, we used the Sgc8, which binds specifically to PTK7 (He, Duan, et al., ^2023a; Sicco et al., ²⁰²⁰), to retarget tumor cells. The SpaCC‐SN3‐Aptamer (SpaCC‐SN3‐Ap) was synthesized using the click reaction of SpaCC‐SN3 with the DBCO‐aptamer. SpaCC‐SN3‐Ap was also visualized by Coomassie blue staining, and the result showed that a protein‐DNA complex (SpaCC‐SN3‐Ap) was successfully constructed (Figure 4c). Anti‐CD3 monoclonal antibody (OKT3) can bind to TCR complexes and activate T cells shifting from a quiescent state to a proliferative and activated state (Hogquist, 2016). Therefore, OKT3 is a commonly used T‐cell‐based immunotherapy. The construction of the fragment crystallizable (Fc) domain of OKT3 (OKT3‐Fc) was the same as our previous study (Figure 4d) (Yang et al., 2023). In addition, the SpaCC‐SN3‐Ap is bound to OKT3‐Fc to form an Ab‐DNA complex (Ab‐SpaCC‐SN3‐Ap) for tumor immunotherapy.

FIGURE 4.

Construction of SpaCC‐SN3‐Aptamer complex. (a) The expression of PTK7 in Jurkat, Nalm6, and Ramos cells was confirmed by western blotting. (b) The binding of the Sgc8 aptamer to Jurkat, Nalm6, and Ramos cells was detected by flow cytometric assay. Ctrl: Sgc8 reverse complement sequence with fluorescence of 6‐FAM. (c) The SpaCC‐SN3‐Ap coupled protein was verified using Coomassie blue staining. (d) Purified OKT3‐Fc protein was identified using Coomassie blue staining.

To determine whether Ab‐SpaCC‐SN3‐Ap complexes, such as OKT3‐SpaCC‐SN3‐Ap, could be used in immunotherapy effectively (Figure 5a), we tested whether OKT3‐SpaCC‐SN3‐Ap could simultaneously bind to T and tumor cells. We stained T and tumor cells with CFSE and APC anti‐human CD19 antibody, respectively, and co‐cultured the mixed (1:1) cells with OKT3‐SpaCC‐SN3‐Ap, OKT3‐Fc, or SpaCC‐SN3‐Ap. If OKT3‐SpaCC‐SN3‐Ap promotes the combination of T and tumor cells, an increased population of FITC‐and APC double‐positive cells could be detected by flow cytometry. The proportion of double‐positive cells in the Nalm6 increased when OKT3‐SpaCC‐SN3‐Ap was present (Figure 5b), suggesting that OKT3‐SpaCC‐SN3‐Ap successfully connected T cells with tumor cells. To exclude the cytotoxicity of OKT3‐SpaCC‐SN3‐Ap on tumor cells, we treated tumor cells with a high concentration of OKT3‐SpaCC‐SN3‐Ap (300 nM) for 24 h and demonstrated that OKT3‐SpaCC‐SN3‐Ap alone did not affect the growth of Nalm6, Ramos, or Su‐DHL‐4 tumor cells (Figure 5c). Next, we examined the effect of OKT3‐SpaCC‐SN3‐Ap on the killing function of T cells at an effector cell: tumor cell (E: T) of 1:1. With the increase in OKT3‐SpaCC‐SN3‐Ap concentration, the killing effect of T cells on PTK7⁺ Nalm6 cells was enhanced, while there was no difference between PTK7⁻ Ramos in OKT3‐Fc and OKT3‐SpaCC‐SN3‐Ap (Figure 5d). In addition, we explored the effects of different E: T ratios at the same OKT3‐SpaCC‐SN3‐Ap concentration. As shown in Figure 5e, the enhanced killing effects of T cells on Nalm6 existed at any E:T ratio. These data indicated that Ab‐SpaCC‐SN3‐Ap could assist T cells in targeting and killing tumor cells effectively.

FIGURE 5.

Ab‐SpaCC‐SN3‐Ap complex mediates the killing function of T cells in vitro. (a) Schematic diagram of targeted killing of tumor cells by OKT3‐SpaCC‐SN3‐Ap. (b) The binding of T cells and tumor cells mediated by OKT3‐SpaCC‐SN3‐Ap was analyzed by flow cytometry. Ctrl: no drug treatment. Scatterplot of flow analysis on the left; double‐positive cell population statistics on the right. (c) Tumor cells were incubated with 300 nM OKT3‐SpaCC‐SN3‐Ap for 24 h. Cell viability was detected by DAPI staining. Ctrl: no drug treatment. (d) Tumor cells were co‐cultured with T cells at E: T = 1:1 ratio for 24 h after addition of 10, 25, 50, and 100 nM SpaCC‐SN3‐Ap, OKT3‐Fc, and OKT3‐SpaCC‐SN3‐Ap. Cell viability was detected by DAPI staining. (e) Tumor cells were co‐cultured with T cells at different E: T ratios for 24 h after the addition of 50 nM SpaCC‐SN3‐Ap, OKT3‐Fc, and OKT3‐SpaCC‐SN3‐Ap. Cell viability was detected by DAPI staining. Ctrl: no drug treatment.

To further elucidate whether the killing effect of T cells could be increased by the OKT3‐SpaCC‐SN3‐Ap complex, we co‐incubated T cells with tumor cells at an E: T ratio of 0.5:1 for 20 h, and measured T‐cell activation markers, such as CD69 and CD25 and T‐cell cytotoxic factors, such as TNF‐α, Granzyme B, and perforin. Flow cytometric analysis revealed that the expression of CD69 and CD25 in CD4⁺ and CD8⁺ T cells was higher in the OKT3‐SpaCC‐SN3‐Ap complex group than in the other groups (Figure 6a,b). Similar results were observed in the secretion of cytotoxic factors (Figure 6c,d). Altogether, these findings suggest that the Ab‐SpaCC‐SN3‐Ap complex specifically stimulates T cell activation and cytokine secretion, thereby enhancing the antitumor effects of T cells.

FIGURE 6.

Ab‐SpaCC‐SN3‐Ap complex boosts the activation of T cells in vitro.(a,b) The expression of T‐cell activation markers, such as CD69 and CD25 was determined in CD4⁺ (a) and CD8⁺ (b) T cells. Ctrl: no drug treatment.(c,d) The expression of T‐cell cytotoxic factors, such as TNF‐α, Granzyme B and perforin was measured in CD4⁺ (c) and CD8⁺ (d) T cells. Ctrl: no drug treatment.

3. CONCLUSION

Antibody drugs are widely used in targeted therapies and immunotherapy, and are also used in protein degradation within targeted cells. Immunotherapies such as BsAbs or Chimeric Antigen Receptor T‐Cell Immunotherapy (CAR‐T) have achieved significant therapeutic effects in most tumor patients, but treatment failure still occurs due to tumor immune escape (Shin et al., 2021). In this study, we took advantage of the C domain of Spa to specifically bind to the Fc segment of IgG to construct the SpaCC, which couples a versatile modified SpyTag with fluorescein, cell‐penetrating peptide, toxins, and aptamers to form the SpaCC‐S‐X protein complex. To obtain the multifunctional characteristics of targeted antigens or cells, SpyTag‐FITC and SpyTag‐TAT were conjugated with SpaCC to form an Ab complex to detect the target cells and deliver antibodies to cells. Based on these properties, SpaCC‐SN3‐MMAE was bound with mAbs to generate an Ab‐drug complex for targeted therapies. Furthermore, SpaCC‐SN3‐Aptamer was connected with the OKT3‐Fc to yield an Ab‐DNA complex for T cell‐based immunotherapy. In conclusion, we designed a multifunctional tool that can be used for targeted therapies and provides a novel approach to drug therapeutics. The SpaC and SpyCatcher‐SpyTag systems can be used to construct a wide range of drugs for the treatment of different diseases, thereby providing a versatile and convenient drug development strategy.

4. MATERIALS AND METHODS

4.1. Materials and chemicals

RPMI‐1640, DMEM, Opti‐MEM™, and CTS™AIMV™ SFM cell culture media were purchased from Gibco(USA). Fetal bovine serum (FBS) was purchased from BioVision(USA). Penicillin–streptomycin (P/S) and 0.25% trypsin–EDTA were purchased from NCM Biotech (China). The PBS, Coomassie brilliant blue, D‐PBS, Imidazole, Ni‐IDA‐Sefinose™ Resin 6FF (Settled Resin), and all DNA used in the selection were purchased from Sangon Biotech (China). Anti‐human CD3 and CD28 antibodies were purchased from SinoBiological (China). The Interleukin‐2 (IL‐2) was purchased from JSSH (China). anti‐rabbit PTK7 Polyclonal antibody or anti‐mouse Beta Actin Polyclonal antibody were purchased from Proteintech (China). MaxFB, MaxFA6, and HEK293 MaxD media were purchased from Mediumbank Biotechnology (China).

4.2. Cell culture

Ramos and SU‐DHL‐4 Cells (from human Burkitt's lymphoma), Jurkat cells (from human acute T‐lymphoblastic leukemia), and Nalm6 cells (from human acute B‐lymphoblastic leukemia) were obtained from the American Type Culture Collection (ATCC). Human embryonic renal epithelial cells (HEK293T) and breast cancer cells (SK‐BR‐3 and MDA‐MB‐231) were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). All cells were cultured in RPMI‐1640 or DMEM medium with 10% FBS and 1% P/S, at 37°C with 5% CO₂.

T cells from healthy volunteers were cultured in CTS™ AIMV™ medium supplemented with 4% fetal bovine serum and 300 IU/mL of human IL‐2. All human samples were obtained with informed consent following the guidelines of the Ethics Committee of Shanghai Children's Medical Center and the Declaration of Helsinki.

4.3. Coomassie blue staining

Protein samples with loading buffer were heated at 95°C for 5 min, and samples were added to a pre‐made 12.5% polyacrylamide gel prepared using the kit (Epizyme Biotech). The gel was electrophoresed in SDS‐PAGE running buffer (Sangon Biotech) at 90 V constant voltage for 30 min and 120 V constant voltage electrophoresis for 60 min. The gel was then washed with water and placed in Coomassie Brilliant blue G‐250 (Beyotime Biotech) for 1 h. Finally, the gel was washed with water and placed overnight in a protein decolorization solution on a shaker. Detection of protein expression using the ChemmiDoc™ MP imaging system (BIO‐RAD, USA).

4.4. Protein expression and purification

The expression vector for the SpaCC protein was transfected into competent BL21 (DE3) E. coli cells (Sangon Biotech). Positive clones were cultured in LB liquid medium containing 10 μg/mL Kanamycin (Sangon Biotech) at 37°C until the OD600 reached 0.6. The IPTG (Sangon Biotech) was added to the culture to 1 mM. Then, the E. coli continued to be cultured at 16°C overnight. Bacterial cells were collected by centrifugation at 3000 rpm for 5 min. After cracking bacterial cells by ultrasonic wave, the soluble fraction was then harvested by centrifugation at 12,000g, 4°C for 30 min for purification.

In addition, HEK293T cells were transfected with a His‐tagged protein expression vector (OKT3‐Fc‐His), Stably expressed cells were screened with 2 μg/mL puromycin and cultured in 15 cm dishes with protein‐producing HEK293 MaxD medium (plus 0.3% MaxFB medium and 3% MaxFA6 medium). The medium was collected every 4–5 days.

To purify the proteins, the obtained bacterial fluid or medium was centrifuged and loaded onto a Ni‐IDA 6FF agarose‐filled purification resin. Proteins were eluted by adding 2 mL of imidazole (20 mM, 40 mM, 0.2 M, 0.5 M, and 1 M). The eluted products were concentrated by centrifugation at 4000 rpm for 30 min at 4°C by Amicon® Ultra‐4 10 K centrifugal filter device (MERCK, USA). The Proteins were analyzed by Coomassie blue staining and protein was stored at 4°C for immediate use or −80°C for long‐term storage.

4.5. Protein conjugation

Purified proteins, synthesized peptides, and aptamers (Tables S1 and S2, all synthesized by Sangon Biotech). SpaCC was coupled with SpyTag‐N3 to construct SpaCC‐SN3 in a 1:2 ratio, and SpaCC was also coupled with SpyTag‐TAT to form SpaCC‐S‐TAT in a 1:10 ratio at 4°C overnight. Similarly, SpaCC was conjugated with SpyTag‐FITC to form SpaCC‐S‐FITC in a 1:2 ratio at 4°C overnight. Ultrafiltration was performed to remove the unreacted peptides. SpaCC‐S‐FITC was then mixed with rituximab or trastuzumab at a 2:1 ratio to form complex CD20/HER2‐SpaCC‐S‐FITC. Finally, SpaCC‐SN3 was coupled with DBCO‐MMAE to form SpaCC‐SN3‐MMAE in a 1:8 ratio at 37°C for 2 h, and ultrafiltration was performed to remove unreacted DBCO‐MMAE. SpaCC‐SN3‐MMAE and Rituximab or Trastuzumab were mixed in a 2:1 ratio to form CD20/HER2‐SpaCC‐SN3‐MMAE; SpaCC‐SN3 was conjugated with DBCO‐aptamer in a 1:4 ratio to form SpaCC‐SN3‐Ap, and OKT3‐Fc was mixed with SpaCC‐SN3‐Ap in a 1:1 ratio to form the Ab‐SpaCC‐SN3‐Ap complex. Coomassie blue staining was used to detect coupling effects.

4.6. Enzyme‐linked immunosorbent assay (ELISA)

We used 1 μg/100 μL Rituximab or Trastuzumab antibody coated in 96‐well plates, overnight at 4°C. Then PBST (PBS with 0.1% tween 20) washed plates to remove free antibodies, and 1.5% BSA was added for 1.5 h at 37°C. After being washed by PBST, SpaCC protein was added according to the Ab: SpaCC molar ratios of 1:0.01, 1:0.1, 1:1, and 1:10, incubated at 37°C for 2 h, Secondary antibodies HRP‐conjugated Anti‐HIS mouse monoclonal antibody (Sangon Biotech) was added and incubated at 37°C for 30 min, After washed by PBST, HRP Chromogen Solution (Beyotime Biotech) was added, and the absorbance was measured at 650 nm.

4.7. Confocal microscopy imaging

Antibody (PE anti‐mouse CD45.1 or FTIC anti‐mouse CD45.2 antibody) (BD Biosciences) was mixed with SpaCC, SpyTag TAT, or SpaCC‐S‐TAT in a ratio of 1:10 at 37°C for 2 h to form the complex. 5 × 10⁵ Jurkat or Ramos cells were resuspended in 600 μL medium, and the complex was added into the medium and incubated at 37°C for 48 h. Then, the cells was washed with PBS, and cell nuclei were stained using 1:2000 Hoechst (Beyotime Biotech) at room temperature for 20 min. Extra dye was washed away with PBS, and the cells were added to a 35‐mm Dish with a 10‐mm Bottom well (Cellvis), detected using a laser scanning confocal microscope (Lecia).

4.8. Western blotting

Ramos, Nalm6, and Jurkat cells (about 10⁶) were lysed in 30 μL SDS Lysis Buffer and the mixture was denatured by heating at 100°C for 20 min and chilling at 25°C. Next, 10 μL of denatured samples were added to the gel wells, and the gel was electrophoresed in Tris‐Glycine SDS‐PAGE Running Buffer (Sangon Biotech) at 90 V constant voltage for 30 min and 120 V constant voltage electrophoresis for 60 min. Next, proteins in SDS‐PAGE gels were transferred to NC membranes using 1 × Rapid Transfer Buffer (Beyotime Biotech) at 400 mA constant current for 30 min, and the NC membrane was blocked by 5% skim milk for 1 h at 25°C. Finally, the NC membrane was incubated overnight at 4°C with primary antibody anti‐rabbit PTK7 Polyclonal antibody or anti‐mouse Beta Actin Polyclonal antibody (Proteintech). The next day, the NC membrane was washed by PBST (PBS containing 1% Tween‐20). Then, the secondary antibody anti‐rabbit IgG HRP‐linked antibody or anti‐mouse IgG HRP‐linked antibody (cell signaling technology) were incubated for 1 h at room temperature. Protein expression was detected using the ChemmiDoc™ MP imaging system.

4.9. Detection of cell expression and binding by flow cytometry

For detection of CD20 and HER2 cells target expression, cells were incubated with or without FITC anti‐human HER2 or APC anti‐human CD20 antibodies (Biolegend) at 4°C incubated for 30 min. Cells were washed and the DAPI (Beyotime Biotech) solution was added to a final concentration of 0.05 μg/mL and detected using BD FACS Canto Plus flow cytometer (USA).

To verify the binding of the Ab‐SpaCC‐S‐FITC complex or aptamer to the target cells, we incubated the 1 × 10⁵ Ramos, SU‐DHL‐4, Jurkat, Nalm6, HEK293T, SK‐BR‐3, and MDA‐MB‐231 cells with or without the addition of CD20/HER2‐SpaCC‐S‐FITC at 37°C for 30 min. SpaCC‐S‐FITC was used as a control, and the cells were detected using a flow cytometer. Similarly, the binding of sgc8 aptamers to cells was evaluated. Cells (about 10 (Rigi et al., 2019)) were suspended in 200 μL binding buffer, then aptamer was added and incubated at 37°C for 30 min. The cells were washed twice or analyzed by flow cytometry, and the data were analyzed using FlowJo v.10.

4.10. In vitro cytotoxicity studies

Nalm6 or Ramos cells (about 10 (Rigi et al., 2019)) were plated on 96‐well plates with 100 μL 1640 medium. Each group was treated with different concentrations of CD20‐SpaCC‐SN3‐MMAE in 0, 1.25, 2.5, 5, 10, 15, 20, 30, 40, and 80 nM. Similarly, HEK293T or SK‐BR‐3 cells (about 10 (Deis et al., 2014)) were treated with a total volume of 100 μL DMEM medium on 96‐well plates. Each group was treated with different concentrations of HER2‐SpaCC‐SN3‐MMAE in 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 nM. After 48 or 72 h at 37°C, 10 μL of CCK8 (Beyotime Biotech) was added, and the cells were incubated for an additional 1 h. The absorbance of the cells was measured at 450 nm by using a microplate reader.

Cell viability was calculated using the following formula: Cell viability (%) = [(As − Ab)/(Ac − Ab)] × 100%. represents the absorbance of the drug‐treated cells. where Ac represents the absorbance value of untreated cells, and Ab represents the absorbance of blank wells (containing an equal volume of medium).

4.11. Cell cycle and apoptosis analysis

For cell cycle analysis, tumor cells were treated with or without drugs for 48 h. After treatment, cells were ruptured using eBioscience™ Foxp3/Transcription (NovoBiotechnology) at 4°C for 30 min. After successful membrane rupture, the cells were washed and centrifuged. Next, the FITC anti‐human Ki‐67 antibody (Biolegend) was added to label the nuclear protein. The cells were incubated at 4°C for another 30 min, washed, and centrifuged. Finally, 0.05 μg/mL of DAPI was added to label the cell cycle, which was detected using flow cytometry.

Cell apoptosis was performed in two groups: HEK293T or SK‐BR‐3 cells treated with 1 nM concentration of the drug for 48 h, followed by trypsin digestion, PBS washing, Annexin V staining (Biolenged) for 30 min at room temperature, and DAPI solution at a final concentration of 0.05 μg/mL. Suspension Nalm6 or Ramos cells were inoculated in 96‐well plates, treated with (1, 10, 100 nM) or no drug for 48 h, and stained with DAPI at a final concentration of 0.05 μg/mL for analysis of apoptosis.

4.12. T cells draw closer to tumor cells

T cells were resuspended in binding buffer and stained with CFSE dye 0.15 μM for 15 min. Nalm6 and Ramos tumor cells were selected for labeling with an APC anti‐human CD19 (Biolenged), incubated at 4°C for 30 min, and then washed with binding buffer. T‐cells and stained tumor cells were mixed at a 1:1 ratio, resuspended with the culture medium with or without the addition of the complexes (OKT3‐SpaCC‐SN3‐Ap, OKT3‐Fc, or SpaCC‐SN3‐Ap), incubated at 37°C for 1 h, and analyzed by flow cytometry.

4.13. Flow cytometry to detect killing

Similarly, tumor cells were labeled with CFSE dye (0.15 μM), and 1 × 10⁵ tumor cells and a certain number of T cells were calculated by E: T was added to each well of a 96‐well plate containing 200 μL of solution with or without complexes (SpaCC‐SN3‐Ap, OKT3‐Fc, OKT3‐SpaCC‐SN3‐Ap) for 24 h. The cells were removed and the co‐incubated cells were transferred to flow tubes. DAPI solution (0.05 μg/mL) was added and detected by flow cytometry.

4.14. T‐cell activation and cytokines assessed by flow cytometry

T cells were co‐cultured with tumor cells in 24‐well plates at a ratio of 0.5:1 with or without the addition of SpaCC‐SN3‐Ap/OKT3‐Fc/OKT3‐SpaCC‐SN3‐Ap at a final concentration of 50 nM for 20 h. After washing with PBS, T cells were labeled with PE‐cy7 anti‐human CD4 (Biolenged) and APC‐cy7 anti‐human CD8 (Biolenged), followed by assessing T cell activation using PE anti‐human CD69 (Biolenged) and Percpcy5.5 anti‐human CD25 (Biolenged) via flow cytometry at 4°C for 30 min.

For cytokine detection, a Golgi blocker (BD Bioscience) was added 6 h before the assay to block intracellular transport. Cells were stained with PE‐cy7 anti‐human CD4 and APC‐cy7 anti‐human CD8 at 4°C for 30 min. After membrane staining, cells were fixed and permeabilized using the Cell Fixation/Cell Cycle™Fixation/Permeabilization Kit (BD Biosciences), then stained with PE anti‐human TNF‐α (BD Biosciences), FITC anti‐human granzyme B (BD Biosciences), and APC anti‐human perforin (BD Biosciences). Finally, the cells were washed, and flow cytometry was performed using FlowJo v.10.

4.15. Statistical analysis

All data were expressed as the mean ± standard deviation (SD). Statistical analyses were performed using the data from three or more individual samples. Graphical analysis was performed using the GraphPad Prism software (version 9.0). Differences between two independent samples were analyzed using unpaired t‐tests, whereas differences between three or more groups were analyzed using One‐way ANOVA. p < 0.05 (*) p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****) indicates a significant difference.

AUTHOR CONTRIBUTIONS

Cai‐Wen Duan: Writing – review and editing; project administration; funding acquisition; resources. Xiu‐Song Huang: Writing – original draft; formal analysis; data curation; conceptualization; validation. Li‐Ting Yang: Validation; data curation; visualization. Ke Yang: Investigation; formal analysis. Hang Zhou: Software; methodology. Tuersunayi Abudureheman: Investigation; data curation. Wei‐Wei Zheng: Investigation; validation. Kai‐Ming Chen: Writing – review and editing; project administration; methodology; supervision; funding acquisition.

Supporting information

Table S1: Protein and peptide sequences.

Table S2: Aptamer sequences.

Figure S1. A coupling of SpyTag‐FITC and SpaCC demonstrated by in‐gel fluorescence.

(A) Detection of SpaCC‐S‐FITC proteins using dual fluorescent channels. The blue band is Marker, and the green band is SpaCC‐S‐FITC protein.

(B) The SpaCC and SpaCC‐S‐FITC proteins were verified by Coomassie blue staining.

(C) The combined photograph of (A) and (B).

Figure S2. Comparison of labeling efficiency between standard antibodies and Ab‐SpaCC‐S‐FITC.

(A) Anti‐human CD20 antibody at a concentration of 0.25 μg, CD20‐SpaCC‐S‐FITC at a concentration of 5 μg. Ctrl: 5 μg SpaCC‐S‐FITC.

(B) FITC‐conjugated Rabbit anti‐human IgG antibody at a concentration of 1 μg, HER2‐SpaCC‐S‐FITC at a concentration of 3 μg. Ctrl‐1: 3 μg SpaCC‐S‐FITC. Ctrl‐2: 1 μg FITC‐conjugated Rabbit anti‐human IgG antibody.

Figure S3. Ab‐SpaCC‐S‐TAT delivers antibodies into target cells.

(A,B) The Ab‐SpaCC/Ab‐SpyTag‐TAT/Ab‐SpaCC‐S‐TAT complexes were co‐incubated with Ramos and Jurkat cells for 24 h by flow cytometry analysis. Ab is PE anti‐mouse CD45.1 (A) or FITC anti‐mouse CD45.2 antibody.  (B), Ctrl: PE anti‐mouse CD45.1 or FITC anti‐mouse CD45.2 antibody.

Figure S4. A coupling of SpaCC‐SN3 or SpaCC‐SN3‐MMAE proteins.

(A) The SpaCC‐SN3 coupled protein was verified by Coomassie blue staining.

(B) The SpaCC‐SN3‐MMAE coupled protein was verified by Coomassie blue staining.

Figure S5. Ab‐SpaCC‐SN3‐MMAE complex blocks the cell cycle of tumor cells in vitro.

(A,B) Tumor cells were incubated with 20 nM CD20‐SpaCC‐SN3‐MMAE (A) or 1 nM HER2‐SpaCC‐SN3‐MMAE (B) for 48 h. The cell cycle of tumor cells was detected by DAPI and Ki‐67 staining. Ctrl: no drug treatment.

Huang X‐S, Yang L‐T, Yang K, Zhou H, Abudureheman T, Zheng W‐W, et al. Construction of a versatile fusion protein for targeted therapy and immunotherapy. Protein Science. 2024;33(4):e4944. 10.1002/pro.4944