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Published in final edited form as: Arch Biochem Biophys. 2023 Dec 5;751:109849. doi: 10.1016/j.abb.2023.109849

Rational Design of Humanized Antibody Inhibitors for Cathepsin S

Po-Wen Yu 1,†, Guoyun Kao 1,†, Zhefu Dai 1, Fariborz Nasertorabi 2, Yong Zhang 1,3,4,5,*
PMCID: PMC10872949  NIHMSID: NIHMS1951017  PMID: 38061628

Abstract

Cathepsin S (CTSS) is involved in pathogenesis of many human diseases. Inhibitors blocking its protease activity hold therapeutic potential. In comparison to small-molecule inhibitors, monoclonal antibodies capable of inhibiting CTSS enzymatic activity may possess advantageous pharmacological properties. Here we designed and produced inhibitory antibodies targeting human CTSS by genetically fusing the propeptide of proCTSS with antibodies in clinic. The resulting antibody fusions in full-length or antigen-binding fragment format could be stably expressed and potently inhibit CTSS proteolytic activity in high specificity. These fusion antibodies not only demonstrate a new approach for facile synthesis of antibody inhibitors against CTSS, but also represent novel anti-CTSS therapeutic candidates.

Keywords: antibodies, cathepsin S, inhibitors, protease, protein engineering

Graphical Abstract

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Introduction

Cathepsin S (CTSS) is a lysosomal cysteine protease present in both intracellular and extracellular space. Unlike other members of the cathepsin family, CTSS remains catalytically active in a broad range of pH and features a relatively restricted expression profile with high levels in spleen and lung [1–7]. Phylogenetically, it belongs to the cathepsin-L like subfamily. CTSS plays important roles in antigen presentation mediated by major histocompatibility complex class II (MHC class II), matrix remodeling, cancer cell growth, and inflammation [5, 8]. It is mostly found in antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs) and also secreted by endothelial cells, smooth muscle cells, epithelial cells, and neutrophils [9].

Dysregulated CTSS expression and proteolytic activity are strongly implicated with many human diseases such as cancer, Alzheimer’s disease, cardiovascular diseases, and autoimmune diseases [5, 8, 10–14]. Overexpressed CTSS is observed in tumors of brain, breast, colon, and pancreas [7, 13]. Through degradation of the invariant chain in MHC class II binding site for antigen presentation, CTSS in tumor microenvironment promotes inactivation of cytotoxic CD8+ T cells and expansion of immunosuppressive CD4+ regulatory T cells (Treg) [7, 15–17]. In addition, CTSS proteolytic activity regulates processing of the invariant chain of MHC class II in B cells and DCs [1, 15, 18, 19]. Thus, CTSS has emerged as an attractive therapeutic target [20].

To block the pathological enzymatic activity of CTSS, various types of small-molecule inhibitors against CTSS have been developed [8, 21–24]. None of them has yet to receive approvals for clinical use, likely due to limited efficacy and/or safety concerns. In comparison to small-molecule agents, monoclonal antibodies are characterized by high affinity and specificity. Moreover, the versatile immunoglobulin scaffolds enable incorporation of new functions and modulation of pharmacological properties [25–38]. Antibody-derived inhibitors of CTSS may provide additional benefits in clinic such as half-life extension, efficacy enhancement, tissue specificity improvement, and toxicity reduction. While anti-CTSS antibodies could be readily prepared, few antibodies with potent inhibition activity specific for CTSS are available [39–41].

We recently developed an approach for facile synthesis of inhibitory antibodies with excellent potency and specificity for cathepsin B (CTSB) and L (CTSL) by genetically fusing their propeptides with clinically approved antibodies [42, 43]. Grafting the propeptide of procathepsin B (proCTSB) onto the heavy chain complementarity determining region 3 (CDR3H) of an anti-human epidermal growth factor receptor 2 (HER2) antibody Herceptin not only transforms the immunoglobulin molecule into a CTSB-specific inhibitor, but also extends the plasma half-life of the propeptide [42, 44]. Attachment of the propeptide of procathepsin L (proCTSL) to the light chain N-terminus of a humanized anti-respiratory syncytial virus (RSV) F protein antibody Synagis results in a potent inhibitory antibody against CTSL [43]. Similar to propeptides of proCTSB and proCTSL, the propeptide of procathepsin S (proCTSS) functions as a potent inhibitor of CTSS [44–46]. Here we extend this approach to generate antibody inhibitors of CTSS through genetic fusions of the propeptide of proCTSS into the CDR3H of full-length Herceptin IgG (designated as Her-HC-CTSSpp IgG) or the N-terminus of the light chain of the fragment antigen-binding (Fab) of Synagis (designated as Syn-LC-CTSSpp Fab) (Fig. 1) [47–51]. The resulting IgG and Fab fusion antibodies revealed selective inhibition activities toward CTSS at low nanomolar levels, representing new agents for anti-CTSS therapy.

Figure 1.

Figure 1.

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Rational design of humanized inhibitory antibodies for CTSS. (A) Generation of a full-length antibody inhibitor of CTSS (Her-HC-CTSSpp IgG) by grafting the propeptide of proCTSS into CDR3H of the anti-HER2 antibody Herceptin. (B) Synthesis of an antibody Fab inhibitor of CTSS (Syn-LC-CTSSpp Fab) by fusing the propeptide of proCTSS to the N-terminus of light chain of the Fab of the anti-RSV F protein antibody Synagis. The propeptide of proCTSS is in red (PDB ID: 2C0Y). The coiled coil-based stalk is in blue. Herceptin and Synagis immunoglobulin scaffolds are in grey (PDB IDs: 1N8Z, 3D6G, and 2HWZ).

Results and Discussion

While anti-CTSS antibodies could be generated through animal immunization or in vitro library-based panning, it remains challenging to identify monoclonal antibodies that are capable of directly engaging with the CTSS catalytic site to inhibit its protease activity. Given our previous successes in designing inhibitory antibodies of CTSB and CTSL by utilizing their propeptides and the fact that the propeptide of proCTSS is an endogenous inhibitor of CTSS [42, 43], we envisioned that functionally grafting the propeptide of proCTSS onto humanized immunoglobulin scaffolds may lead to potent antibody inhibitors with high specificity for CTSS.

To this end, the propeptide of proCTSS (Q17-R113) was attempted for replacement of CDR3H (W99-M107) of the anti-HER2 antibody Herceptin (Fig. 1A) [47–49]. To facilitate folding of the propeptide within the antibody variable region, a previously established coiled coil-based stalk motif that supports functional CDR fusions was utilized [25, 26, 29]. In addition to the Herceptin IgG-based antibody inhibitor (Her-HC-CTSSpp IgG), we designed a Fab antibody inhibitor (Syn-LC-CTSSpp Fab) by fusing the propeptide to the N-terminus of the Fab light chain of the anti-RSV F protein antibody Synagis (Fig. 1B) [50, 51]. In comparison to the full-length antibody inhibitor, the Fab antibody inhibitor is much smaller and carries no fragment crystallizable (Fc) domain, possibly giving rise to unique physicochemical and pharmacological properties to meet distinct biomedical research needs [52]. Unlike the Herceptin scaffold, the Synagis is expected to recognize no human antigens and therefore allows for fusion of the propeptide at its light chain N-terminus, which may reduce impact of the fused propeptide on folding of the immunoglobulin.

The designed Herceptin full-length heavy chain CDR fusion and Synagis light chain N-terminal fusion were constructed by overlap extension PCR and cloned into mammalian expression vectors. In combination with the vector encoding Herceptin light chain or Synagis heavy chain Fab, the sequence-verified fusion vectors were used to express Her-HC-CTSSpp IgG and Syn-LC-CTSSpp Fab antibody fusions through transient transfection of Expi293F cells. Herceptin IgG and Synagis Fab antibodies were also expressed in the transiently transfected Expi293F cells by using the same type of expression vectors harboring native heavy and light chains.

The expressed antibodies were purified by protein G affinity chromatograph. In comparison to the yield of Herceptin IgG (5.5 mg/L), the yield of Her-HC-CTSSpp IgG decreased to 0.6 mg/L. Different from the IgG antibody CDR3H fusion, the Synagis Fab N-terminal fusion could be expressed in a yield (14.4 mg/L) comparable to that of native Synagis Fab (15 mg/L). SDS-PAGE gel analysis showed that the light chains of Herceptin IgG and Her-HC-CTSSpp IgG remained at the same position, whereas the heavy chain with the fused propeptide migrated at around 75 kDa, much higher than that of the Herceptin heavy chain and consistent with the design (Fig. 2). Similarly, the light chain of Syn-LC-CTSSpp Fab shifted up to near 45 kDa relative to that of Synagis Fab on the basis of SDS-PAGE gels. And the heavy chains of both Fab antibodies migrated at the same molecular weight (Fig. 2). Consistent with the measured expression yields, thermal shift assays showed significantly decreased melting temperature (Tm) for Her-HC-CTSSpp IgG (74.8 ± 0.1°C) in comparison with that of Herceptin IgG (88.2 ± 0.1°C) and similar Tm values for Syn-LC-CTSSpp Fab (82.2 ± 0.1°C) and Synagis Fab (84.5 ± 0.1°C) (Fig. S1). Gel filtration chromatography revealed slight aggregates for Her-HC-CTSSpp relative to Herceptin IgG, Synagis Fab and Syn-LC-CTSSpp Fab (Fig. S2 and S3). These results indicate that while the CDR3H fusion of propeptide reduces stability of Herceptin, the N-terminally fused propeptide has slightly minor effects on folding of the Synagis Fab antibody.

Figure 2.

Figure 2.

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Coomassie-stained SDS-PAGE gel analysis of purified antibodies.

The inhibition activity of Her-HC-CTSSpp IgG was then examined with human CTSS through fluorescence-based activity assays using a fluorogenic peptide substrate Z-VVR-AMC (Fig. S4A). Importantly, the designed Her-HC-CTSSpp IgG could potently inhibit human CTSS in a dose-dependent manner (Fig. 3A). By fitting the measured initial reaction rates into the competitive inhibition equation, the calculated Ki of Her-HC-CTSSpp IgG for CTSS is 6.03 ± 0.52 nM (Fig. 3B). CTSS and CTSL belong to the same subfamily of cathepsins. And the propeptide of proCTSS is a known inhibitor of CTSL [45, 46]. Therefore, the activity of Her-HC-CTSSpp IgG in inhibiting human CTSL was evaluated with a fluorogenic peptide substrate Z-FR-AMC (Fig. S4B). The fluorescence-based signals indicated that human CTSL could be inhibited by Her-HC-CTSSpp IgG at nanomolar levels with a Ki value of 49.33 ± 13.70 nM (Fig. 3C and D). The parental antibody scaffold Herceptin displays no inhibition activity for human CTSS or CTSL at a concentration of 1000 nM. To further study inhibition specificity of Her-HC-CTSSpp IgG, fluorescence-based activity assays for human CTSB and bovine trypsin were carried out in the absence or presence of Her-HC-CTSSpp IgG. No inhibitory effects on CTSB or trypsin protease activity were observed for Her-HC-CTSSpp IgG at concentrations up to 1000 or 500 nM (Fig. 3E and F). These results demonstrate that grafting the propeptide of proCTSS into Herceptin CDR3H creates a potent and selective inhibitor for human CTSS.

Figure 3.

Figure 3.

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Inhibition potency and specificity of Her-HC-CTSSpp IgG. (A) Inhibition activity of Her-HC-CTSSpp IgG for human CTSS. (B) Determined Ki of Her-HC-CTSSpp IgG for human CTSS. (C) Inhibition activity of Her-HC-CTSSpp IgG for human CTSL. (D) Determined Ki of Her-HC-CTSSpp IgG for human CTSL. (E) Effects of Her-HC-CTSSpp IgG on human CTSB proteolytic activity. (F) Effects of Her-HC-CTSSpp IgG on bovine trypsin proteolytic activity. Her-HC-CTSSpp IgG at various concentrations were incubated with human CTSS, CTSL, CTSB, or bovine trypsin in the presence of their respective fluorogenic substrates for up to 20 minutes. Proteolytic activities were monitored based on measured real-time fluorescence intensities. PBS and Herceptin were included as controls.

Next, inhibition potency and specificity of the designed Syn-LC-CTSSpp Fab antibody were analyzed by fluorescence-based proteolytic activity assays. Similar to Her-HC-CTSSpp IgG antibody, Syn-LC-CTSSpp Fab exhibits potent inhibitory activity toward human CTSS with a determined Ki of 2.12 ± 0.19 nM, slightly lower than that of Her-HC-CTSSpp IgG (Fig. 4A and B). Likewise, Syn-LC-CTSSpp Fab functions as a weaker antibody inhibitor against human CTSL. In comparison to CTSS, its potency for CTSL (Ki: 24.51 ± 4.11 nM) decreases by about 12-fold (Fig. 4C and D). Other inhibition specificity assays with human CTSB and bovine trypsin indicated that Syn-LC-CTSSpp Fab displays no inhibitory effects on these protease activities at concentrations up to 500 nM (Fig. 4E and F). As controls, Synagis Fab antibody at a concentration of 500 nM shows no inhibition activities for CTSS, CTSL, CTSB, or trypsin. Taken together, these results support the generation of a potent Fab antibody inhibitor with high specificity for human CTSS.

Figure 4.

Figure 4.

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Inhibition potency and specificity of Syn-LC-CTSSpp Fab. (A) Inhibition activity of Syn-LC-CTSSpp Fab for human CTSS. (B) Determined Ki of Syn-LC-CTSSpp Fab for human CTSS. (C) Inhibition activity of Syn-LC-CTSSpp Fab for human CTSL. (D) Determined Ki of Syn-LC-CTSSpp Fab for human CTSL. (E) Effects of Syn-LC-CTSSpp Fab on human CTSB proteolytic activity. (F) Effects of Syn-LC-CTSSpp Fab on bovine trypsin proteolytic activity. Syn-LC-CTSSpp Fab at various concentrations were incubated with human CTSS, CTSL, CTSB, or bovine trypsin in the presence of their respective fluorogenic substrates for up to 20 minutes. Proteolytic activities were monitored based on measured real-time fluorescence intensities. PBS and Synagis were included as controls.

This work demonstrates facile generation of potent inhibitory antibodies with high specificity for CTSS. Guided by structure analysis, two different types of antibody inhibitors targeting human CTSS were successfully generated by genetically fusing the propeptide of proCTSS into two distinct immunoglobulin scaffolds. Both the full-length IgG and Fab antibody fusions could inhibit CTSS protease activity at low nanomolar levels (Ki: 6.03 nM for Her-HC-CTSSpp IgG and 2.21 nM for Syn-LC-CTSSpp Fab), comparable to that of the recombinant propeptide of proCTSS (Ki: 2.50–7.60 nM) measured at a similar pH level in previous studies [44, 45]. The inhibition potency of Her-HC-CTSSpp IgG and Syn-LC-CTSSpp Fab for human CTSS is 8–12-fold higher than that for human CTSL. In contrast, the propeptide of proCTSS shows less than 6-fold selectivity for CTSS over CTSL [44, 45]. These results suggest that attachments of the propeptide onto IgG and Fab molecules have little effects on its inhibition activity for CTSS and the parental antibody scaffolds may slightly enhance specificity of the propeptide against CTSS.

In comparison to previous designs that produced only IgG-based inhibitors for CTSB or CTSL [42, 43], this work develops both IgG and Fab antibody inhibitors against CTSS, a protease important for antigen processing. Like the previously generated Herceptin-based CTSB antibody inhibitor, CDR3H fusion-derived Her-HC-CTSSpp IgG is expected to display no binding toward HER2 receptor and improved plasma stability for the propeptide. The Synagis-based Fab inhibitor likely features lack of binding to other human antigens. While these antibody inhibitors are anticipated to block extracellular CTSS proteolysis, they may be internalized via Fc receptors or membrane bound protease molecules to inhibit intracellular CTSS activity. As conformation of the propeptide is likely to be pH dependent and neutral pH was shown to increase inhibition of human CTSS by the propeptide [46], the CTSS antibody inhibitors may exhibit higher inhibition activities for extracellular CTSS in a more neutral environment than intracellular CTSS under acidic conditions.

Importantly, these studies establish genetic fusion of cathepsin propeptide into immunoglobulins as a facile approach for developing inhibitory antibodies targeting cathepsins. In contrast to conventional library-based screening methods, utilizing the propeptides for rational design of antibody inhibitors is more efficient in identifying candidates with desired inhibition functions. Furthermore, the resulting propeptide-containing antibodies could be engineered by modulating adjacent interacting loops for enhanced affinity and/or specificity. In addition, bi- and multi-functional antibody inhibitors could be developed for targeting different types of proteases or diseased tissues. Future studies for these inhibitory antibodies along with the propeptide include measurements of in vitro and in vivo stability, characterization of inhibition activity at varied pH conditions, improvement of potency and selectivity, and assessment of cellular uptake, immunogenicity, and biological and pharmacological activity with cellular and animal models of CTSS-associated diseases.

Conclusions

Humanized antibody inhibitors in both IgG and Fab formats for human CTSS were generated by engineering clinically approved antibodies with the propeptide of proCTSS. These antibody inhibitors reveal potent and selective activities against CTSS, providing new biologic agents for targeting CTSS proteolytic activity.

Materials and Methods

Materials.

All synthetic DNA fragments and polymerase chain reaction (PCR) primers were purchased from integrated DNA technologies (Coralville, IA). DNA restriction enzymes NheI (R3131S), EcoRI (R3101S), and T4 DNA ligase (M0202S) were purchased from New England Biolabs (Ipswich, MA). Recombinant human CTSS (1183-CY-010), human CTSL (952-CY-010), human CTSB (953-CY-010), and fluorogenic peptide substrate Z-FR-AMC (ES009) were purchased from R&D Systems (Minneapolis, MN). Fluorogenic peptide substrate Z-VVR-AMC (BML-P199–0010) and Boc-QAR-AMC (BML-P237–0005) were purchased from Enzo Life Sciences (Farmingdale, NY). Bovine trypsin (T1426) and centrifugal filter units (UFC801008) were purchased from Sigma-Aldrich (St.Louis, MO). AccuPrime Pfx DNA polymerase (12344024), Zeocin (R25001), and Expi293F expression system (A14635) were purchased from Thermo-Fisher Scientific (Waltham, MA). Protein G resin (L00209), Tris-MOPS-SDS running buffer powder (M00138), and ExpressPlus-PAGE gels (M42015) were purchased from GenScript (Piscataway, NJ). DNA gel recovery kits (D4001), DNA clean & concentrator kits, and plasmid maxiprep kits (D4202) were purchased from Zymo Research (Irvine, CA).

Molecular cloning.

The synthetic gene encoding the propeptide of proCTSS (Q17-R113) was purchased from integrated DNA Technologies and amplified by PCR with AccuPrime Pfx DNA polymerase with the forward and reverse primers. The amplified CTSS propeptide was confirmed by DNA gel electrophoresis and then purified by DNA gel extraction kits. To generate the Herceptin IgG-based antibody inhibitor (Her-HC-CTSSpp IgG), a flexible GGGGS linker was added at each end of the propeptide. The entire sequence was then fused with the coiled coil-based stalk at each end in replacement of Herceptin CDR3H (W99-M107) via overlap extension PCR. The sequences of the coiled coil-based stalk are: H2N-GGSGAKLAALKAKLAALK-COOH in N-terminus and H2N-ELAALEAELAALEAGGSG-COOH in C-terminus. To create the Synagis Fab-based antibody inhibitor (Syn-LC-CTSSpp Fab), the amplified DNA fragment of propeptide was fused to the light chain N-terminus of Synagis with a GGGGS linker by overlap extension PCR. The purified fusion genes were digested with restriction enzymes EcoRI and NheI and ligated into the pFuse vector backbone by T4 DNA ligase. Electrocompetent DH10B cells were then transformed by the resulting ligation products via electroporation. The sequences of the resulting expression constructs were verified by DNA sequencing (Genewiz, CA).

Antibody expression and purification.

Herceptin IgG, Her-HC-CTSSpp IgG, Synagis Fab, and Syn-LC-CTSSpp Fab were expressed through transient transfection of Expi293F cells with respective heavy and light chains. The Expi293F cells were grown at 37°C with 5% CO2 in the Expi293F expression medium. Culture media were harvested on day 5 post transfection and then centrifuged at 4,000 g for 30 minutes. The expressed antibodies were purified with protein G resins by eluting with 100 mM glycine (pH 2.7) and neutralizing with 1/10 volume of 1 M Tris-HCl (pH 8). The purified antibodies were buffer exchanged with PBS in centrifugal filters and analyzed by SDS-PAGE gels.

Thermal shift assays.

Thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl) phenyl] maleimide (CPM) (Thermo Fisher Scientific, catalog no. D346) was used for stability profiling [53]. CPM was dissolved in DMSO at a final concentration of 4 mg mL−1. A 40× dilution of the dye in PBS was used as a working solution.

Each protein sample (5–10 μg) in doublets was diluted in PBS to a final volume of 95 μL, and thereafter 5 μL of 40× CPM was added to achieve a final volume of 100 μL. Samples were mixed properly and incubated in the dark for 15 minutes before they were transferred to a QIAGEN’s real-time PCR cycler, Rotor-Gene Q (Qiagen. Inc, USA) to be heated in a controlled manner with a ramp rate of 0.2°C second−1. The assays were performed over a temperature range of 25°C – 95°C. The excitation and the emission wavelengths were set at 365 nm and 460 nm, respectively.

The generated data was processed using the Rotor-Gene Q software series to calculate melting temperature (Tm). The first derivative of the fluorescence signal over temperature (dF/dT) was plotted against T, and the melting temperature was calculated for each sample as the peak maximum. The graphs were generated from the raw data by GraphPad Prism 9 (San Diego, CA).

Gel filtration chromatography.

Synagis Fab or Syn-LC-CTSSpp Fab (150 μg each) was injected onto a Superdex 75 Increase 10/300 GL column (GE Healthcare Life Sciences, Pittsburgh, PA). Herceptin IgG or Her-HC-CTSSpp IgG (150 μg each) was loaded onto a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences, Pittsburgh, PA). Each antibody construct was eluted with PBS (pH 7.4). Fraction samples were analyzed via Coomassie-stained SDS-PAGE gels.

Michaelis constants of fluorogenic substrates.

To determine Km values of fluorogenic substrates Z-Val-Val-Arg-AMC (Z-VVR-AMC) and Z-Phe-Arg-AMC (Z-FR-AMC) for human CTSS and CTSL, respectively, Z-VVR-AMC and Z-FR-AMC were prepared as 10 mM stocks in DMSO. Following dilution with assay buffers (CTSS: 50 mM NaOAc, 250 mM NaCl, 5 mM DTT, pH 5; CTSL: 400 mM NaOAc, 4 mM EDTA, 8 mM DTT, pH 5.5), Z-VVR-AMC or Z-FR-AMC at various concentrations was incubated with human CTSS (1 ng/μL) or CTSL (0.1 ng/μL) in assay buffer at 25°C. The fluorescence intensities were recorded by a Synergy H1 Hybrid Multi-Mode reader for 10 minutes (excitation: 380 nm; emission: 460 nm). The initial rates of CTSS or CTSL proteolytic activity were analyzed to determine Km of Z-VVR-AMC or Z-FR-AMC by GraphPad Prism 9 (San Diego, CA).

Enzyme inhibition assays.

To measure inhibition activities against CTSS for Her-HC-CTSSpp IgG and Syn-LC-CTSSpp Fab, 25 μL of human CTSS with a working concentration of 1 ng/μL in assay buffer (50 mM NaOAc, 250 mM NaCl, 5 mM DTT, pH 5) was incubated with 25 μL of Her-HC-CTSSpp IgG or Syn-LC-CTSSpp Fab at various concentrations and followed by the additions of 50 μL of fluorogenic substrate Z-VVR-AMC at a concentration of 100 μM.

To determine inhibition activities toward CTSL for Her-HC-CTSSpp IgG and Syn-LC-CTSSpp Fab, human CTSL was first diluted into 40 μg/mL in assay buffer (400 mM NaOAc, 4 mM EDTA, 8 mM DTT, pH 5.5) and incubated on ice for 15 minutes. Human CTSL (25 μL at a working concentration of 0.2 ng/μL in assay buffer) was then added with 25 μL of Her-HC-CTSSpp IgG or Syn-LC-CTSSpp Fab at various concentrations and 50 μL of fluorogenic substrate Z-FR-AMC at a concentration of 160 μM.

To evaluate inhibition activities on CTSB for Her-HC-CTSSpp IgG and Syn-LC-CTSSpp Fab, human CTSB was diluted into 10 μg/mL in assay buffer (34 mM NaOAc, 60 mM acetic acid, 4 mM EDTA, 5 mM DTT, pH 5.5) and incubated at room temperature for 15 minutes. Human CTSB (25 μL at a working concentration of 0.1 ng/μL in assay buffer) was mixed with 25 μL of Her-HC-CTSSpp IgG or Syn-LC-CTSSpp Fab at various concentrations and 50 μL of fluorogenic substrate Z-FR-AMC at a concentration of 160 μM.

To assess inhibition activities with bovine trypsin for Her-HC-CTSSpp IgG and Syn-LC-CTSSpp Fab, bovine trypsin was dissolved with 1 mM HCl into 1 mg/mL and then prepared into 10 nM in assay buffer (PBS, pH 7.4). Bovine trypsin (25 μL at a working concentration of 10 nM in assay buffer) was incubated with 25 μL of Her-HC-CTSSpp IgG or Syn-LC-CTSSpp Fab at various concentrations and 50 μL of fluorogenic substrate BOC-QAR-AMC at a concentration of 60 μM.

The fluorescence intensities (excitation: 380 nm; emission: 460 nm) for above assays were measured by a Synergy H1 Hybrid reader at 25°C. The inhibition constant Ki was calculated by fitting initial reaction rates and inhibitor concentrations to the competitive inhibition equation: (V’0/V0) = (Km + [S])/(Km + [S] + (Km[I]/Ki)), where V’0 is the initial reaction rate with inhibitor, V0 is the initial reaction rate without inhibitor, [I] is the inhibitor concentration, and [S] is the substrate concentration.

Supplementary Material

1

  • Design of inhibitors of cathepsin S by exploiting its propeptide and antibodies.

  • Antibody-propeptide fusions potently inhibit cathepsin S.

  • Generation of IgG- and Fab-based antibody inhibitors specific of cathepsin S.

Funding

G.K. acknowledges the financial support from the USC Taiwan Global Fellowship. This work was supported in part by Sharon L. Cockrell Cancer Research Fund, National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) grant R35GM137901 (to Y. Z.), National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the NIH grant R01EB031830 (to Y. Z.), and National Cancer Institute (NCI) of NIH grant R01CA276240 (to Y. Z.).

Footnotes

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Supporting Information

Thermal shift assays and gel filtration chromatography of antibody constructs and determined Michaelis constants of fluorogenic peptide substrates.

Declaration of competing interest

The authors declare no conflict of interest.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1951017-supplement-1.docx (842.8KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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