Background: Proteolytic impairment of the Fc effector functions of therapeutic monoclonal antibodies (mAbs) can compromise their antitumor efficacy in the tumor microenvironment and may represent an unappreciated mechanism of host immune evasion. Pertuzumab is a human epidermal growth factor receptor 2 (HER2)-targeting antibody and has been widely used in the clinic in combination with trastuzumab for treatment of HER2- overexpressing breast cancer. Pertuzumab susceptibility to proteolytic hinge cleavage and its impact on the drug’s efficacy has not been previously studied. Methods: Pertuzumab was incubated with high and low HER2-expressing cancer cells and proteolytic cleavage in the lower hinge region was detected by western blotting. The single hinge cleaved pertuzumab (scIgG-P) was purified and evaluated for its ability to mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro and anti- tumor efficacy in vivo. To assess the cleavage of trastuzumab (IgG-T) and pertuzumab (IgG-P) when simultaneously bound to the same cancer cell surface, F(ab’) fragments of IgG-T or IgG-P were combined with the intact IgG-P and IgG-T, respectively, to detect scIgG generation by western blotting. Results: Pertuzumab hinge cleavage occurred when the mAb was incubated with high HER2-expressing cancer cells. The hinge cleavage of pertuzumab caused a substantial loss of ADCC in vitro and reduced antitumor efficacy in vivo. The reduced ADCC function of scIgG-P was restored by an anti-hinge mAb specific for a cleavage site neoepitope. In addition, we constructed a protease-resistant version of the anti-hinge mAb that restored ADCC and the cell-killing functions of pertuzumab when cancer cells exressed a potent IgG hinge-cleaving protease. We also observed increased hinge cleavage of pertuzumab when combined with trastuzumab. Conclusion: The reduced Fc effector function of single hinge-cleaved pertuzumab can be restored by an anti- hinge mAb. The restoration effect indicated that immune function could be readily augmented when the damaged primary antibodies were bound to cancer cell surfaces. The anti-hinge mAb also restored Fc effector function to the mixture of proteolytically disabled trastuzumab and pertuzumab, suggesting a general therapeutic strategy to restore the immune effector function to protease-inactivated anticancer antibodies in the tumor microenvironment. The findings point to a novel tactic for developing breast cancer immunotherapy. Keywords: Pertuzumab, HER2, Antibody hinge cleavage, Fc effector function, Breast cancer, Tumor invasion of humoral immunity * Correspondence: Ningyan.email@example.com; firstname.lastname@example.org Equal contributors Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, the University of Texas Health Science Center at Houston, 1825 Pressler St., Suite 532, Houston, TX 77030, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hsiao et al. Breast Cancer Research (2018) 20:43 Page 2 of 12 Background of basic biology, and suggests an application of that basic Previous studies have indicated that pathogen-associated biological information to clinical situations in which poly- and tumor-associated proteases are capable of cleaving clonal auto-antibodies are present against in situ tumor human IgG1 within or adjacent to the hinge region [1–6]. associated antigens (TAA). To this point, we investigated For example, a group of tumor-associated proteases such whether an AHA was effective at targeting both hinge as matrix metalloproteinase MMP3, MMP7, MMP9, and cleaved IgG-T and IgG-P in combination to restore ADCC MMP12 generate limited cleavage of human IgG1 in vitro, and antitumor activity. Taken together, our results suggest and in some cases demonstrably in vivo. Such cleavage that using AHA to restore anticancer immunity is a prom- can confer substantial functional impairment to thera- ising strategy for developing a new class of breast cancer peutic antibodies [2, 4, 6]. In addition to F(ab’) fragments immunotherapy. with their Fc domains removed, IgG1 antibodies with a single proteolytic cleavage in the lower hinge region Methods (scIgG1), but with the Fc domain remaining attached, also Cell culture and reagents exhibit impaired antibody-dependent cell-mediated cyto- All cancer cell lines were obtained from the American toxicity (ADCC) and complement-dependent cytotoxicity Type Culture Collection (ATCC, Manassas, VA, USA) (CDC) [6–8]. We have demonstrated this susceptibility and maintained as previously described [7, 9]. Trastuzu- for trastuzumab in clinical tumor samples as shown with mab was obtained from a specialty pharmacy as previ- detection of single hinge-cleaved trastuzumab (scIgG-T) ously described . Pertuzumab with a single proteolytic in tumor tissues from patients with breast cancer treated cleavage in the lower hinge region (ScIgG-P) was pre- with trastuzumab as neoadjuvant . pared in house using a specific hinge cleavage proteinase In related investigations, it was shown that anti-hinge (IdeS) (Sigma-Aldrich, St Louis, MO, USA). Intact IgG-P antibodies (AHAs) that specifically bind to the neoepitope and protease-resistant IgG-P (PRIgG-P) were con- formed by enzymatic scission successfully restored Fc- structed based on variable sequences of pertuzumab, dependent function to cleaved therapeutic antibodies [7, expressed in HEK293F cells, and purified using Protein 8, 10]. Polyclonal AHAs purified from human intravenous A affinity chromatography as previously described . immunoglobulin (IVIG) was shown to restore function to Isotype control antibodies used in the study were pre- a set of antigen-specific therapeutic monoclonal anti- pared using the same expression system and protocols bodies disabled by proteolytic hinge cleavage . In a sep- as the HER2 targeting IgG-P antibodies. arate study, we were able to demonstrate strong ADCC restoration of scIgG-T by a monoclonal AHA . In a Preparation of scIgG-P and F(ab’) fragments model system using the potent IdeS protease (expressed Both scIgG-T and scIgG-P were prepared using IdeS par- by S. pyogenes), AHAs were also found to be subject to tial cleavage by monitoring the disappearance of intact proteolytic attack in the hinge region with a resulting loss IgG using non-reducing SDS-PAGE detection. After the of restorative capability . To address this issue, we ap- partial cleavage of IgG hinge, the mixtures of scIgGs and plied a protein engineering approach to derive a protease- F(ab’) fragments were separated using Protein A agarose resistant monoclonal antibody (mAb). This version of an (ThermoFisher, Waltham, MA, USA) to elute the bound otherwise proteolysis-susceptible mAb retained the re- scIgGs and free Fc fragment from unbound F(ab’) . Then quired Fc function in protease-rich environments [7, 11]. CaptureSelect™ Kappa XL Affinity Matrix (ThermoFisher) Pertuzumab (IgG-P) is a humanized mAb targeting was used to further purify F(ab’) fragments from the flow human epidermal growth factor receptor 2 (HER2)  through of the protein A purification step, while in a sep- at an epitope different from that of trastuzumab (IgG-T) arate step the free Fc fragment from Protein A elution was [13, 14]. Specifically, IgG-P interacts with domain II of removed to enrich scIgGs from the CaptureSelect™ Kappa HER2 whereas IgG-T targets domain IV of the HER2 re- XL affinity Matrix. The purity of both scIgG and the ceptor [13, 14]. It has been reported that ADCC is an F(ab’) was > 95% as shown on fast protein liquid chroma- important IgG-P mechanism of action [15–20]. There tography (FPLC) size exclusion chromatography. have been no reports of any previous study of IgG-P sus- ceptibility to proteolytic hinge cleavage. IgG-P single-hinge cleavage when bound to a cancer cell In this study, we demonstrated the occurrence of hinge line cleavage of IgG-P in cell cultures and that the scission of a SKBR3, BT474, MCF7-HER2, and MCF7 breast cancer single peptide bond in this region diminished the anti- cells and SKOV3 ovarian cancer cells were seeded on 6- tumor activity and ADCC functions of IgG-P. We also well plates at 80% confluence and incubated for 24 h. The found enhanced hinge cleavage for HER2-bound IgG-P cancer cells were treated with 10 μg/ml of IgG-T, IgG-P, when combined with trastuzumab. The latter observation IgG-T F(ab’) ,orIgG-P F(ab’) for designated periods. Cells 2 2 pointed to a conceptual model to incorporate observations were harvested and lysed using radioimmunoprecipitation Hsiao et al. Breast Cancer Research (2018) 20:43 Page 3 of 12 assay (RIPA) buffer (ThermoFisher) containing a 10% pro- NJ, USA) subcutaneously (sc.) at the hind-leg fat pad to tease inhibitor cocktail (ThermoFisher). Monoclonal anti- establish tumors as we described previously . BT474 bodies and F(ab’) fragments were enriched using Protein A breast cancer cells (5 × 10 cells/mouse) were implanted (ThermoFisher) and their concentrations were determined into 6 to 8 week old mice and antibody treatment was as previously described . Briefly, Protein A magnetic initiated after one additional week. The mAb treatments beads were incubated with cell lysates at 4 °C for 1 h, and were performed once a week by intraperitoneal (ip) the captured antibodies were collected in SDS containing injection for 5 weeks at a dosage of 10 mg/kg body sample buffer (Bio-Rad). Samples were subjected to SDS- weight. Tumor growth and mouse health were monitored PAGE and WB detection using a goat anti-human Fc-HRP twice per week. Tumor growth was quantified by conjugate (1:4000) (Jackson Immune Research Laboratory, measuring the size of tumors using a Vernier scale caliper. West Grove, PA, USA) as previously described [7, 9]. Purification of human anti-hinge cleavage site antibodies Detection of HER2 expression in breast cancer cell lines from Octagam (IVIG) by flow cytometry Abiotinylatedhuman IgG1 hinge peptide analogue with The cancer cells were detached using non-enzymatic solu- the sequence biotin-THTCPPCPAPELLG (peptide 1981B) tion (Fisher Scientific) from a cell culture flask and or a biotinylated IgG-P F(ab’) fragment (generated with blocked in PBS buffer with 1% BSA for 45 min at room the IdeS protease) were used as the absorbents to isolate temperature. IgG-P was used to stain HER2 and R-PE human anti-hinge cleavage site autoantibodies from IVIG (phycoerythrin) conjugated F(ab’) goat anti-human IgG (pooled, purified IgGs from human plasma). The IVIG was Fcγ (1:200) (Jackson Immune Research Laboratory) was diluted in PBS to a protein concentration of 1 mg/ml and used as detection antibody. For the determination of the was incubated with streptavidin agarose beads with bound anti-hinge antibody binding to scIgG-P and scIgG-T on peptide 1981B or biotinylated IgG-P F(ab’) for 1 h at 4 °C cancer cell surfaces, AHA (mAb 2095–2) was biotinylated followed by three washes with PBS. Bound antibodies were and the binding of the AHA was detected using R-PE con- eluted with 50 mM glycine (pH 2.6) then neutralized by jugated streptavidin (1:200) (Jackson Immune Research la- adding 1/10th volume of 1 M Tris (pH 8.0). The antibody boratories). All stained cells were analyzed by a Guava eluent was exchanged into PBS by adding 10× volume of easyCyte HT flow cytometer according to the manufac- PBS and concentrated using Amicon centrifugal filter units turer’s instructions (Millipore, Hayward, CA, USA). (MWCF, 30 kDa) (Millipore). Specificity enrichment of AHAP- F(ab’) was also performed by running the eluent Detection of CD4, CD8 and CD56 expression level in human through an additional affinity step with intact IgG-P linked peripheral blood mononuclear cells (PBMCs) cells by flow on agarose. The flow through from the second enrichment cytometry step was buffer exchanged and concentrated using Amicon CD4, CD8, and CD56 positive cells in PBMCs isolated from centrifugal filter units (MW, 30 kDa) (Millipore). healthy human donors were detected by flow cytometry on a fluorescence-activated cell sorter FACScan (Becton Dick- Antibody-dependent cellular cytotoxicity (ADCC) assay inson, Walpole, MA, USA). Alexa Fluor 700 anti-CD4 Polyclonal human AHAs and the monoclonal AHA (eBioscience, San Diego, CA, USA), anti-CD8-Per-CP-Cy5. (2095–2) were examined for their ability to restore ADCC 5, and anti-CD56-Per-CP-Cy5.5 (BD Pharmingen, San activity using a non-invasive gold microelectrode-based Diego, CA, USA) antibodies were used to detect expression cell cytotoxicity assay by the xCELLigence instrument levels of CD4, CD8, and CD56, respectively. Approximately (ACEA Biosciences, San Diego, CA, USA) as described 1× 10^ pelleted PBMC cells were blocked in PBS buffer previously . SKOV3 and SKBR3 cancer cells were used with 1% BSA for 20 min at room temperature. The cells as target cells (T) and human PBMCs, freshly isolated were then stained with antibodies at 4 °C for 30 min, from two healthy donors, were used as effector cells (E) washed twice in PBS buffer with 1% BSA and resuspended with the E:T ratio at 25:1. The degree of ADCC restor- in 0.5 ml staining buffer for FACScan analysis. ation by AHA coupled with scIgG-P was by comparison to the cells treated with IgG-P (30 nM), or scIgG-P Mouse xenograft tumor model (30 nM), respectively, with or without AHA (60 nM). The All animal procedures and care were conducted in accord- ADCC rescuing efficacy of polyclonal human AHAs or ance with the animal care and use guidelines and the monoclonal AHA (2095–2 mAb) was measured by adding protocol was approved by the Animal Welfare Committee scIgG-P alone or in combination with scIgG-T together (AWC) of the University of Texas Medical School at with a twofold to tenfold excess of AHAs. The percentage Houston. Breast cancer cells (BT474) with high HER2 ex- of cell lysis was defined as: (cell index of control group – pression were prepared and implanted into athymic nude cell index of treatment group)/cell index of control group) nu + mice (Foxn1 /Fox1 genotype, Envigo, East Millstone, × 100. All experiments were replicated three times (n =3). Hsiao et al. Breast Cancer Research (2018) 20:43 Page 4 of 12 ELISA for assessing antibody binding to antigen HER2 occurs on the cell surfaces when IgG-P engages its HER2 A microtiter plate (ThermoFisher) was pre-coated with antigen target rather than in solution. recombinantly expressed human HER2 extracellular do- main protein (SinoBiological, Beijing, China) at 2 μg/ml Single hinge cleavage impeded the anti-tumor function of overnight at 4 °C in PBS. Microtiter wells were washed IgG-P with PBS and blocked with 200 μl/well of 3% BSA in PBS It has been reported that ADCC is an important mechan- for 1 h at room temperature. Serial dilutions of IgG-P, ism in the anticancer efficacy of pertuzumab . To test PRIgG-P, or F(ab’) fragments were compared with the in- whether proteolytic hinge cleavage of pertuzumab results in tact IgG-T/IgG-P antibodies for binding after incubating a loss of Fc-mediated cell killing function, we compared for 1 h at room temperature. After washing with PBS measurements of ADCC activity mediated by scIgG-P and (three times), goat anti-human Fc-specific HRP conjugate intact IgG-P. We used a highHER2expressingSKOV3 (ThermoFisher) (1:4000) was used for detection with 3,3'- ovarian cancer cell line as the target and freshly isolated 5,5' tetramethylbenzidin (TMB) (ThermoFisher) for PBMCs as immune effector cells. The group treated with 10 min incubation. The reaction was stopped by adding scIgG-P had significantly less lysis of cancer cells than the 50ul/well of 1 N H SO and the individual wells were read group treated with intact IgG-P (Fig. 2a). To compare 2 4 for absorbance at 450 nm using a plate reader (Spectra- scIgG-P antitumor function with the intact IgG-P in vivo, Max M4, Molecular Devices, Sunnyvale, CA, USA). we adopted a murine xenograft tumor model in which mice were inoculated with an established high HER2-expressing Statistical analysis cell line. Seven days after subcutaneous implantation of the The pair-wise Student t test was used for statistical ana- cancer cells, tumor-bearing micewererandomlydivided lysis using GraphPad software. Statistical significance into groups (n = 5) for treatment with scIgG-P or IgG-P at was defined as a p value ≤0.05. a dose of 10 mg/kg, once weekly for five weeks. In addition to the isotype control IgG, IgG-P-N297A (IgG-P with a sin- Results gle amino acid mutation at position 297 to limit glycosyla- Detection of IgG-P hinge cleavage when incubated with tion of IgG-P) was used as a control group for a loss of Fc high HER2-expressing cancer cells function. In comparison with the isotype control, all three As part of the ongoing investigation into whether antibody pertuzumab antibody versions - scIgG-P, the N297A mu- hinge cleavage represents a meaningful occurrence for tant, and intact IgG-P - inhibited tumor growth, but both IgG1 anticancer mAbs, we tested the hinge cleavage of scIgG-P and N297A mutant were significantly less effective pertuzumab (IgG-P) during incubation with high HER2- than the intact IgG-P (Fig. 2b). With regard to the aglycosy- expressing cancer cells. As illustrated in Fig. 1a, the anti- lated N297A mutant of IgG1, it has been established that body with a single hinge cleavage (scIgG1) can be resolved this variant confers reduced Fc-mediated immune cell en- into four components after separation by SDS-PAGE: light gagement and decreased ADCC due to impairment of Fc chain, full length heavy chain, hinge-cleaved heavy chain receptor binding . Thus, the comparable reduction of (scHC, upper fragment from the nicked hinge containing tumor volumebyscIgG-P andthe aglycosylatedIgG-P- the Fab domain), and Fc monomer (Fc(m)). There was de- N297A mutant pointed to a related mechanism of immune tectable Fc(m) in cell lysates after a 24-h incubation of impairment (Fig. 2b). Tumor volumes at the end point of IgG-P with high HER2-expressing cancer cells (BT474, the xenograft study for individual mice in the four treat- SKOV3, SKBR3, and MCF7-HER2). IgG-P and scIgG-P ment groups are shown in Fig. 2c. The data further demon- were extracted from the cell lysates using Protein A beads strated that both the scIgG-P and the N297A mutant and hinge cleavage, as indicated by presence of Fc(m), was exhibited significantly less tumor inhibition efficacy than tested by western blotting (WB) analysis using an anti- the intact IgG-P. human Fc-specific detection antibody (Fig. 1b-e,top panels). SKBR3 cancer cells showed much stronger Fc(m) Anti-hinge cleavage site autoantibodies (AHA) rescued generation than the other high HER2-expressing cancer the ADCC activity of scIgG-P cell lines (Fig. 1d, top panel). In contrast, low HER2- In a previous study, human AHAs were purified using expressing MCF7 cancer cells and IgG-P incubated with F(ab’) affinity chromatography . Those purified auto- conditioned medium from cell culture did not have de- antibodies from IVIG restored biological functions to F(ab’) tectable levels of Fc (m) (Fig. 1f, top panel and g). High generated from a variety of monoclonal antibodies. In this HER2 expression in BT474, SKOV3, SKBR3, and MCF7- study, we enriched AHA from IVIG using a peptide HER2 cells (Fig. 1b-e, bottom panels) were detected by analogue of the point of IdeS cleavage of the human IgG1 FACS. In contrast, no HER2 expression was detected in hinge (peptide 1981 sequence ending in PAPELLG- ). COOH MCF7 cancer cells (Fig. 1f, bottom panel). The latter result AHA demonstrated a degree of restoration of the indicates that antibody hinge cleavage preferentially ADCC activity of scIgG-P diminished by the IdeS protease Hsiao et al. Breast Cancer Research (2018) 20:43 Page 5 of 12 b cd ef g Fig. 1 Pertuzumab (IgG-P) hinge cleavage was detected when IgG-P was incubated with higher human epidermal growth factor receptor 2 (HER2)-expressing cancer cell lines but not in low HER2 expressing cancer cell line. a Fragments of IgG with a single proteolytic cleavage in the lower hinge region (scIgG) generated under denaturing and reducing conditions, as assessed by western blotting detection. Fc(m) is the Fc monomer from the hinge cleavage and sc-Heavy chain indicates the N-terminal fragment from the hinge cleavage. Western blots showing hinge cleavage of IgG-P for the cell lines: BT474 (b, top panel); SKOV3 (c, top panel); SKBR3 (d, top panel); and MCF7-HER2, a MCF7 breast cancer cell line overexpressing HER2 (e, top panel). Low levels of Fc(m) were detected in MCF7 cells without HER2 expression (f, top panel). Cells were treated with 10 μg/ml of IgG-P for 4 h and 24 h at 37 °C, 5% CO in serum-free medium. Protein A magnetic beads were used to pull down the IgG-P proteolytic product. The hinge cleavage product, Fc monomer, was visualized by blotting the membrane using a secondary detection antibody, goat anti-human Fc-HRP antibody. A band shown on the western blotting with a molecular weight of 25 kDa was the Fc(m), which was seen in the scIgG-P enzymatically cleaved at the hinge region by immunoglobulin G-degrading enzyme S (IdeS). The intact IgG-P did not show a detect- able band on the western blotting under reduced and denatured gel running conditions. High HER2 expression in BT474 (b, bottom panel), SKOV3 (c, bottom panel), SKBR3 (d, bottom panel), and MCF7-HER2 (e, bottom panel), and no detectable level of HER2 expression in MCF7 cells (f, bottom panel) were measured by FACS. g Detection of IgG-P hinge cleavage in cancer cell culture medium. Cancer cell-conditioned medium from BT474, SKOV3, SKBR3, MCF7, and MCF7-HER2 after treatment with IgG-P were collected after 24-h incubation and subjected to western blotting using a secondary detection antibody, anti-human Fc-HRP antibody: 10 μl of cancer cell-conditioned medium was loaded in each lane (Fig. 3a). For comparison purposes, we also purified AHA restore ADCC activity to scIgG-T [7, 10]. In this study, we from IVIG using IgG-P F(ab’) (generated with IdeS) as the investigated the analogous potential of using AH-mAb absorbent and tested its ability to restore ADCC to F(ab’) . (2095–2) to rescue the function of scIgG-P. Indeed, the As shown in Fig. 3b,AHA showed a comparable AH-mAb 2095–2 was strongly bound to scIgG-P on high P-F(ab’)2 level of ADCC restoration to AHA .Inpreviousstudies, HER2-expressing cancer cells (Fig. 3c), and as expected, re- a monoclonal antibody AHA (2095–2) was shown to stored the ADCC activity of scIgG-P to a level comparable Hsiao et al. Breast Cancer Research (2018) 20:43 Page 6 of 12 a b Isotype IgG1 71% Ig-G-P ** scIgG-P IgG-P-N297 ** ** 20% 7 1014172124273134 Day post treatment Fig. 2 Single hinge cleavage caused a loss of antibody dependent cellular cytotoxicity (ADCC) activity in intact pertuzumab (IgG-P) that contributed to less tumor inhibition in IgG with a single proteolytic cleavage in the lower hinge region (scIgG-P) treatment group. a ADCC- targeted lysis of SKOV-3 ovarian cancer cells by IgG-P and scIgG-P was examined using the electrode impedance assay. SKOV-3 cells (5000 cells/ well) were seeded on the E-plate as the target cell and peripheral blood mononuclear cells (25,000 cells/well) isolated from a single donor were used as the immune effector cells in complete cell culture medium containing scIgG-P (30 nM) and IgG-P (30 nM). The cell index after 96 h of incubation was the experimental end point (n = 3). The percentage of cell lysis was defined as: (cell index of control group – cell index of treatment group)/cell index of control group) × 100. b Tumor volumes from nude mice (n = 5) were inoculated subcutaneously with 5 × 10 BT474 human breast cancer cells and treated with isotype IgG1 control, IgG-P, scIgG-P, or IgG-P N297A at 10 mg/kg weekly for a total of five doses until tumors reached an average size of 100mm . c Tumor volumes at the end time point of the nude mice xenograft study for individual mice treated with isotype IgG1 control, IgG-P, scIgG-P, and IgG-P N297A. Tumor size was measured twice a week. The error bars in the graphs depict the standard deviation (SD) obtained in three independent experiments. *p < 0.05,**p < 0.01 with that of the intact IgG-P with SKOV3 ovarian cancer the target cell and PBMCs were used as the immune ef- cells (Fig. 3d) or SKBR3 breast cancer cells (Fig. 3e)as the fector cell source. PRIgG-P clearly induced a higher per- target cells. centage of cell lysis (> 60%) than IgG-P (< 20%) (Fig. 4d). Next, weexaminedthe ADCC restorativefunctionofa A variant of IgG-P, engineered to resist protease hinge protease-resistant anti-hinge mAb, PR2095–2, in the IdeS- cleavage, confirmed the impact of local protease action expressing cellular environment.Again,the SKOV3-IdeS on IgG function cell line was used as the target cell and PBMCs were used An engineered Fc variant of trastuzumab (PRIgG-T) was as the source of immune effector cells. SKOV3-IdeS cells previously shown to withstand protease attack and to retain incubated with PR2095–2 and IgG-P had a higher percent- ADCC function in a protease-rich environment compared age of cell lysis (~ 65%) than the group treated with 2095–2 to IgG-T . In this study, we constructed a protease- and IgG-P (< 15%) at the end point of the experiment resistant variant of pertuzumab (PRIgG-P) using the same (96 h) (Fig. 4e). These results indicated a clear benefit of experimental approach. PRIgG-P demonstrated strong re- the engineered protease-resistant hinge for mAb-mediated sistance to IdeS proteolysis compared to IgG-P when incu- ADCC in the IdeS protease-rich environment. bated with the protease-expressing BT474-IdeS and SKOV3-IdeS cells (Fig. 4a). As expected, PRIgG-P had Elevated IgG-P hinge cleavage occurred when IgG-T and similar binding to the antigen HER2 extracellular domain IgG-P were combined (ECD) as IgG-P (Fig. 4b, c). We investigated PRIgG-P IgG-T and IgG-P are often used in combination in patients antibody-mediated ADCC activity in cells with elevated with breast cancer with high HER2 expression. To investi- proteolytic activity. The SKOV3-IdeS cell line was used as gate how the hinge impairment of IgG-T and IgG-P affects Tumor volume (mm ) Hsiao et al. Breast Cancer Research (2018) 20:43 Page 7 of 12 ab cd Fig. 3 Anti-hinge antibodies rescued antibody dependent cellular cytotoxicity (ADCC) activity for single hinge cleaved pertuzumab (scIgG-P). a-b Purified human anti-protease-induced, anti-hinge autoantibodies (AHA) using peptide analogues representing hinge-immunoglobulin G- degrading enzyme S (IdeS) cleavage sites, 1981 or F(ab’) generated by digesting immunoglobulin G (IgG-P) with IdeS as the absorbent, restored ADCC activity for scIgG-P. SKOV-3 cell (5000 cells/well) was seeded on the E-plate as the target cell and peripheral blood mononuclear cells PBMCs (25,000 cells/well) isolated from a single donor were used as the immune effector cell in complete cell culture medium containing scIgG-P (30 nM). The percentage of cell lysis was defined as: (cell index of control group – cell index of treatment group)/cell index of control group) × 100. c Flow cytometry showing binding results for AH-mAb with IgG-P or scIgG-P on surfaces of high human epidermal growth factor receptor 2-expressing cancer cells. Biotinylated 2095–2 and streptavidin-PE conjugate were used for cell staining. d-e 2095–2 ADCC rescuing effect for scIgG-P at varying concentrations. A fixed concentration of 30 nM for IgG-P with threefold dilutions from 30 nM for 2095–2 were used in the ADCC assay. SKOV-3 cells (5000 cells/well) and SKBR3 cell (7000 cells/well) were used as the target cells and PBMCs isolated from a single donor were used as the immune effector cells at an effector (E)-target (T) ratio of 25:1 the combination treatment, we assessed the cleavage of not predicted in advance, the addition of the F(ab’) of antibodies when simultaneously bound to the same cancer IgG-T accelerated the generation of Fc(m) from IgG-P. cell surface. For detection of scIgG generation, F(ab’) This finding is unique in providing evidence for altered fragments of IgG-T or IgG-P were combined with the in- proteolytic kinetics of an antibody in a simultaneous bind- tact IgG-P and IgG-T, respectively. After incubation with ing circumstance. Intriguingly, there was not a corre- either the BT474 or the SKOV3 cancer cell line, any sponding increase in Fc(m) generation from IgG-T when detected Fc(m) must have derived from the intact IgG. combined with the F(ab’) of IgG-P (Fig. 5b). Structural re- This additive test system was made possible by the simi- arrangements have been observed for IgG-T and IgG-P larity in the binding affinity for HER2 ECD between the simultaneously interacting with HER2 ECD in an in silico F(ab’) fragments and the corresponding full-length ver- analysis , which may explain the elevated IgG-P hinge sion of either IgG-P or IgG-T (Fig. 5a). Although it was cleavage in the presence of IgG-T. Hsiao et al. Breast Cancer Research (2018) 20:43 Page 8 of 12 ab c de Fig. 4 Pertuzumab variants with Fc engineered to withstand protease attack, protease-resistant variant of pertuzumab (PRIgG-P) and PR2095–2, restored lost antibody dependent cellular cytotoxicity (ADCC) activity for immunoglobulin G (IgG-P) in an immunoglobulin G-degrading enzyme S (IdeS)-rich environment. a The hinge cleavage profiles are shown for IgG-P, Protease-resistant variant of trastuzumab (PRIgG-T), and PRIgG-P for the SKOV3 ovarian cancer cell line overexpressing the IdeS protease (SKOV3-IdeS), and for the BT474 breast cancer cell line overexpressing IdeS protease- stable cell lines (BT474-IdeS). The SKOV3-IdeS and BT474-IdeS cancer cell lines were treated with 10 μg/ml of IgG-P/PRIgG-P/PRIgG-T for 24 h at 37 °C, 5% CO in serum-free medium. IgG-P and scIgG-P generated by digesting IgG-P with IdeS were used as standards. Protein A magnetic beads were used to pull down the IgG hinge proteolytic products, which were visualized by western blotting. b IgG-P and PRIgG-P binding affinity to human epidermal growth factor receptor 2 (HER2) receptor by ELISA. Microtiter plate wells were coated with recombinant human HER2 extracellular domain (ECD) at a concentration of 2 μg/ml as the antigen. IgG-P/PRIgG-P was used as the primary antibody then detected by goat anti-human Fc-HRP conjugate. c Flow cytometry showing the association between PRIgG-P or IgG-P and HER2 ECD on the cell surface. R-PE conjugated F(ab’) goat anti-human IgG Fcγ was used for detection. d Comparison of ADCC activity between IgG-P and PRIgG-P. SKOV-3-IdeS ovarian cancer cell line (5000 cells /well) was used as the target cell and peripheral blood mononuclear cells (PBMCs) (25,000 cells/well) isolated from a single donor were used as the immune effector cell. The percentage of cell lysis was defined as: (cell index of control group – cell index of treatment group)/cell index of control group)×100. e Comparison of ADCC activity between 2095 and 2 and PR2095–2 in an IdeS-rich environment. The SKOV-3-IdeS cell (5000 cells/well) was used as the target cell and PBMCs (25,000 cells/well) isolated from a single donor were used as the immune effector cells. Fixed concentrations of 30 nM of IgG-P and 60 nM of 2095–2/PR2095–2, respectively, were used in the ADCC assay. Experiments were conducted in triplicate and the error bars in the graphs correspond to SDs obtained in three independent experiments Anti-hinge cleavage site antibodies rescued ADCC activity as the group treated with intact IgG-P and IgG-T at all with a mixture of scIgG-T and scIgG-P time points (Fig. 6b). This indicated that 2095–2 was able To determine whether the AHA can restore ADCC of to access the single hinge cleavage site of the therapeutic scIgG-T and scIgG-P when used together on a HER2- antibodies in combination on the same cell surface and re- expressing cell, we added purified human polyclonal anti- ceptor. These results were extended to an examination of hinge autoantibodies (AHA or AHA ) to a com- the restoration phenomenon in a protease-enriched set- P- F(ab’)2 1981 bination of scIgG-T and scIgG-P. As expected, the com- ting. For this, the IdeS-expressing SKOV-IdeS cell line bination of scIgG-T, scIgG-P, and purified polyclonal was used for the anti-HER2 combination and the parental human AHA produced a higher percentage of cell lysis and the protease-resistant versions of 2095–2 anti-hinge than the target cell line (SKOV3) treated with the combin- mAb were tested for ADCC restoration. In this case, ation of scIgG-T and scIgG-P alone (Fig. 6a). Next, we ex- PR2095–2, but not 2095–2, successfully rescued ADCC amined the ADCC rescuing effect of AH-mAb (2095–2) activity with the combination treatment (Fig. 6c). Thus, for the scIgG-T and scIgG-P combination treatment. The cell lytic functions of combined hinge-cleaved anti-HER2 target cell line (SKOV3) treated with scIgG-P and scIgG-T mAbs were recovered by polyclonal and monoclonal anti- combined with 2095–2 showed a similar level of cell lysis hinge antibodies in multiple settings. Hsiao et al. Breast Cancer Research (2018) 20:43 Page 9 of 12 ab Fig. 5 Intact trastuzumab (IgG-T) and intact pertuzumab (IgG-P) combination treatment increased IgG-P cleavage. aThe binding affinity to human epidermal growth factor receptor 2 (HER2) extracellular domain (ECD) for IgG-P, IgG-T and F(ab’) fragments of IgG-T and IgG-P. Microtiter plate wells were coated with HER2 ECD at a concentration of 2 μg/ml as the antigen. Threefold dilutions of IgG-P, IgG-T and the F(ab’) fragments of IgG-T and IgG-P were each applied to microtiter wells coated with recombinant human HER2 ECD. Goat anti-human kappa light chain-HRP conjugate was used as the detection antibody. b IgG-T and IgG-P proteolytic cleavage profile with/without addition of IgG-P-F(ab’) fragment and IgG-T-F(ab’) fragment, respectively. BT474 2 2 breast cancer cell line or SKOV3 ovarian cancer cell line were treated with IgG-T (10 μg/ml) with/without F(ab’) fragment of IgG-P (10 μg/ml) or vice versa for4hand 24 hat37°C, 5% CO in serum-free medium. Protein A magnetic beads were used to pull down the IgG-P proteolytic product. The hinge cleavage product, Fc monomer, was visualized by blotting the membrane using a secondary detection antibody, goat anti-human Fc-HRP antibody Fig. 6 Anti-hinge cleavage site antibodies rescued antibody dependent cellular cytotoxicity (ADCC) activity for a mixture of single hinge cleaved trastuzumab (scIgG-T) and single hinge cleaved pertuzumab (scIgG-P). SKOV-3 cells (5000 cells/well) were seeded on the E-plate as the target cell and peripheral blood mononuclear cells (25,000 cells/well) isolated from a single donor were used as the immune effector cells in complete cell culture medium containing a mixture of intact pertuzumab (IgG-P) (30 nM) and intact trastuzumab (IgG-T) (30 nM), or scIgG-P (30 nM) and scIgG-T (30 nM) with and without anti-hinge antibody (AHA) (120 nM). The percentage of cell lysis was defined as: (cell index of control group – cell index of treatment group)/cell index of control group)×100. a ADCC activity for a combination of IgG-T and IgG-P (black bar), a combination of scIgG-P and scIgG-T (white bar), and a combination of scIgG-T and scIgG-P using human anti-protease-induced AHA using peptide analogues representing hinge- immunoglobulin G-degrading enzyme S (IdeS) cleavage sites, 1981B (dark gray bar) or F(ab’) generated by digesting IgG-P with IdeS as the absorbent (light gray bar). b ADCC activity for a combination of IgG-T and IgG-P (black bar), a combination of scIgG-P and scIgG-T (white bar), and a combination of scIgG-T and scIgG-P using the anti-hinge mAb 2095–2 (dark gray bar). c ADCC cell lysis of the IdeS-expressing SKOV3-IdeS cell line by a combination of IgG-T and IgG-P (black bar), a combination of IgG-T and IgG-P + anti-hinge mAb 2095–2 (white bar), and a combination of IgG-T, IgG-P, and protease-resistant PR2095–2 (dark gray bar) Hsiao et al. Breast Cancer Research (2018) 20:43 Page 10 of 12 Discussion Consequently, an alternative and well-defined model sys- The susceptibility of IgGs to functional inactivation by pro- tem was considered to be essential for the present study. teolytic enzymes has been studied in various ways including The specificity and potency of IdeS for cleaving the IgG purified systems using cancer-associated enzymes, en- hinge enabled this attempt [6, 24–26]. This was confirmed dogenous proteases expressed by tumor cells, and model by the demonstration that IgG-P was enzymatically cleaved cell lines with enhanced protease secretion. The present in- at the hinge when incubated with IdeS expressing cancer vestigation touched on these aspects as they might relate to cell lines and in the solution-phase. The precise peptide the considerable complexity of the in vivo tumor environ- bond specificity of IdeS in targeting the hinge region of hu- ment and therapeutic approaches used to treat it. man IgGs led to the development or isolation of antibodies Pertuzumab (IgG-P) is often administered to patients that specifically detect the presence of the hinge cleavage with HER2-positive breast cancer together with trastuzu- site. By extension of these findings, it is possible to consider mab (IgG-T) as combination therapy [12, 18, 20, 21]. Both therapeutic options for restoring IgG function by the asso- IgG-P and IgG-T target HER2 but interact with different ciation of a functional anti-hinge IgG to the site of IgG pro- domains of HER2 [13, 14]. The present findings demon- teolysis in cell-bound IgGs. The concept is not limited to strated that there was enhancement of IgG-P cleavage on IdeS and can apply to physiologically relevant, cancer- the cell surface by endogenous proteolytic action when the related proteases in the tumor environment. mAb was used in combination with trastuzumab. In Anti-hinge autoantibodies can be found in healthy indi- addition, the inherent sensitivity of IgG-P to the hinge viduals and patients with inflammatory diseases [5, 27]. cleavage was different from that for IgG-T. Substantial Indeed, purified autoantibodies prepared from serum IgGs levels of hinge proteolysis of IgG-P were detected when using immobilized F(ab’) generated from IdeS-cleaved IgG-P was incubated with SKBR3 cells, while IgG-T had IgG-P as the absorbent or using immobilized peptide pos- lower sensitivity on this high HER2-expressing cancer cell sessing the “…PAPELLG” sequence with the free C- line . In silico data suggest a structural rearrangement of terminal glycine showed modest restoration of ADCC ac- IgG-T and IgG-P when both mAbs are bound to the HER2 tivity to scIgG-P in vitro. These findings support the con- receptor simultaneously . The present finding of inter- cept that endogenous anti-hinge autoantibodies, especially dependent protease susceptibility further extends the topo- at enhanced levels, might be efficacious in certain disease logical dynamics of the receptor. For example, the circumstances. Further, the development of anti-hinge formation of the HER2-pertuzumab complex may cause re- monoclonal antibodies to rescue compromised Fc- arrangement of the receptor-antibody complex to expose mediated functions in hinge-cleaved mAbs is a readily previously inaccessible proteolytic sites buried inside the achievable approach for this purpose [6–8]. The monoclo- antibody protein structure . Structure-based methodolo- nal AHA 2095–2 used in this study targets the neoepitope gies likely will be needed to detail the interactions among of IdeS cleaved IgG  and can restore the ADCC activ- the targeted antigen, therapeutic antibodies, and proteases. ity of scIgG-T in vitro and also the inhibition of tumor Studies have implicated the involvement of Fc-mediated growth by administering scIgGT in vivo [7, 10]. This study ADCC activity in IgG-P-mediated inhibition of tumor demonstrated that AHA 2095–2 restored ADCC activity growth [15, 16, 20]. We earlier showed that the cleavage of of scIgG-P as well. Moreover, mAb 2095–2restored func- a single peptide bond in the hinge caused a partial loss of tion to both scIgG-T and scIgG-P when the two distinct, the ADCC function of IgG-T in vitro and in vivo [7, 9]. In dysfunctional anti-HER2 mAbs were used in combination. this study, we showed a similar reliance on Fc structural in- Thus, these interconnected findings suggest substantial tegrity for IgG-P-mediated ADCC effector function and flexibility for AHA as a therapeutic approach for cancer tumor inhibition in vitro and in vivo. The single hinge treatment. In addition, a promising alternative strategy cleaved IgG-P and an engineered immune cell engagement using an engineered protease-resistant hinge in trastuzu- deficient mutant of pertuzumab (IgG-P N297A) showed mab was capable of overcoming the protease susceptibility decreased tumor inhibition. Our results suggest that IgG-P of the original IgG. In protease-expressing cellular set- with a cleaved hinge partially impedes tumor inhibition due tings, PRIgG-T conferred resistance to proteolytic hinge to the loss of Fc effector function. The partial inhibition of cleavage both in vitro and in vivo . In the present study, tumor growth by scIgG can be attributed to a lack of inter- the concept was applied successfully to pertuzumab and ference with the Fc-independent pathway of pertuzumab to the anti-hinge mAb 2095–2 and suggests broad gener- cell killing via HER2 antigen engagement. ality for this approach within the tumor environment. We and others have reported that MMPs are associated with antibody hinge cleavage in tumor tissues [4, 9]. Nu- Conclusions merous proteases coexist in a tumor microenvironment. This study showed a readily detectable level of IgG-P hinge This poses a hurdle for attributing IgG functional loss to cleavage when incubated with high HER2-expressing breast particular enzymes or mixtures of enzymes . cancer cell lines (but not with low HER2-expressing cells) Hsiao et al. Breast Cancer Research (2018) 20:43 Page 11 of 12 and suggests that IgG proteolysis is facilitated when bound Competing interests The authors declare that they have no competing interests. to the cell surface. ScIgG-P showed substantial loss of ADCC activity compared to un-cleaved IgG-P in vitro and was less potent against tumorgrowthinvivo. Thelossof Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in ADCC activity of scIgG-P can be restored by anti-hinge published maps and institutional affiliations. antibodies. An Fc engineering approach to derive a protease-resistant platform was shown to be applicable in Received: 22 November 2017 Accepted: 26 April 2018 two ways: (1) for directly maintaining IgG-P ADCC func- tion in a protease-rich environment by engineering resist- References ance into the heavy chain of IgG-P and (2) by the indirect 1. Agniswamy J, Lei B, Musser JM, Sun PD. 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Published: Jun 1, 2018
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