Research ArticleExperimental Studies
Open Access

Antibody-dye Conjugates Targeting EGFR and HER2 for the Photoimmunotherapy of Bladder Cancer

FABIAN HUBER, ISIS WOLF, JONAS STORZ, SUSANNE SCHULTZE-SEEMANN, SUSAN LAUW, LUKAS KLEMENZ, ARKADIUSZ MIERNIK, CHRISTIAN GRATZKE, REINHARD BRÜCKNER and PHILIPP WOLF
Anticancer Research May 2024, 44 (5) 1837-1844; DOI: https://doi.org/10.21873/anticanres.16985

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Abstract

Background/Aim: Although there are curative treatment options for non-muscle-invasive bladder cancer, the recurrence of this tumor is high. Therefore, novel targeted therapies are needed for the complete removal of bladder cancer cells in stages of localized disease, in order to avoid local recurrence, to spare bladder cancer patients from stressful and expensive treatment procedures and to increase their quality of life and life expectancy. This study tested a new approach for the photoimmunotherapy (PIT) of bladder cancer. Materials and Methods: We generated a cysteine modified recombinant version of the antibody cetuximab targeting the epidermal growth factor receptor (EGFR) on the surface of bladder cancer cells. Then, we coupled the novel photoactivatable phthalocyanine dye WB692-CB1 via a maleimide linker to the free cysteines of the antibody. PIT was performed by incubating bladder cancer cells with the antibody dye conjugate followed by irradiation with visible red light. Results: The conjugate was able to induce specific cytotoxicity in EGFR-positive bladder cancer cells in a light dose-dependent manner. Enhanced cytotoxicity in RT112 bladder cancer cells was evoked by addition of a second antibody dye conjugate targeting HER2 or by repeated cycles of PIT. Conclusion: Our new antibody dye conjugate targeting EGFR-expressing bladder cancer cells is a promising candidate for the future PIT of bladder cancer patients.

Key Words:

Bladder cancer (BC) is one of the most common types of cancers worldwide with around 573,000 new cases and 213,000 deaths per year (1). In approximately 70% of cases, bladder tumors are still superficially confined to the urothelium or the lamina propria at the time of diagnosis and have not yet invaded the muscle (2). Transurethral tumor resection followed by intravesical chemotherapy with mitomycin C is used as first-line therapy for non-muscle-invasive BC (NMIBC). Gemcitabine is increasingly used as a more cost-effective alternative with comparable efficacy. In patients with an intermediate risk profile, intravesical immunomodulation with Bacillus Calmette-Guérin (BCG) is used to reduce the risk of progression (3, 4). Despite such potentially curative treatment options, the recurrence in NMIBC is high. The recurrence rates for patients with low, intermediate, or high-risk BC are approximately 60%, 70% or 80%, respectively (5, 6). As a result, cost-intensive cystoscopies must be performed regularly after treatment for surveillance. If the tumor progresses and becomes muscle-invasive (MIBC), patients have to undergo complex and stressful therapies including neoadjuvant or adjuvant chemotherapy in combination with radical cystectomy and pelvic lymphadenectomy or partial bladder resection in combination with radio-chemotherapy (4). Patients with metastasized BC receive palliative chemotherapy. Novel immunotherapeutic approaches include the use of checkpoint inhibitors (avelumab, mivolumab, pembrolizumab) or antibody drug conjugates targeting nectin-4 (Enfortumab vedotin) or Trop-2 (Sacituzumab govitecan) (7, 8). Despite such medical advances, patients with metastasized BC have a poor prognosis with a median overall survival of only a few months (9). New and targeted therapeutic approaches are therefore needed for the complete removal of BC cells in stages of localized disease, in order to avoid local recurrence, to spare BC patients from stressful and expensive treatment procedures and to increase their quality of life and life expectancy.

One therapeutic option for the treatment of localized tumor lesions, can be photoimmunotherapy (PIT) that was developed by Mew et al. in 1983 (10). PIT is based on the use of conjugates consisting of a tumor-specific antibody and a photoactivatable dye (photosensitizer). After binding of the antibody dye conjugate to target antigens on the surface of tumor cells, the dye can be activated by light of a suitable wavelength and induces cell death (11). The cell death mechanisms of PIT have not yet been conclusively clarified. Conjugates containing the phthalocyanine dye IR700 were observed to induce a conformational change when bound to the cell surface, ultimately resulting in the disruption of cell membrane integrity (12). Other studies assume the generation of reactive oxygen species followed by lipid peroxidation and membrane leakage (13). PIT has several advantages over conventional tumor therapies. The antibody dye conjugates are highly tumor specific. Moreover, the visible light used for photosensitizer activation is non-ionizing and therefore harmless (11). Therefore, surrounding normal tissue that does not express tumor antigens and/or is not in the light focus is spared from the treatment. Moreover, cell rupture by PIT results in a leakage of damage-associated molecular patterns (DAMPs) and induction of immunogenic cell death (ICD) (11). This means that in addition to local antitumor effects PIT can also induce systemic ones, for example against tumor cells that have already spread or metastasized.

In the present study, we evaluated the PIT in BC cells using conjugates consisting of the antibodies cetuximab or trastuzumab targeting the human epidermal growth factor receptor (EGFR, ERBB1) or the human epidermal growth factor receptor 2 (HER2, ERBB2), respectively, and of the photoactivatable phthalocyanine dye WB692-CB1, that was generated by us.

Materials and Methods

Cell lines. The EGFR and HER2-positive BC cell lines RT112, HT1376 and T24 were obtained from the German Collection of Microorganisms and Cell Cultures (Leibnitz Institute, Braunschweig, Germany). The EGFR and HER2 negative control cell line CHO was purchased from Gibco (Invitrogen, Karlsruhe, Germany). RT112 cells were cultivated in RPMI1640 Medium (Gibco), 10% fetal calf serum (Sigma Aldrich, St. Louis, MO, USA) and penicillin/streptomycin (100 U/ml, 100 mg/l) at 37°C and 5% CO2. HT1376 and T24 cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) medium (Gibco) and CHO cells in F-12 Nutrient Mixture Medium (Gibco) containing the same supplements.

Generation of the anti-EGFR antibody CmbT120C/D265C. The heavy chain genes, encompassing the cysteine mutations T120C and D265C (EU numbering), along with genes of the variable domain of the light chain (VL) of the chimeric anti-EGFR antibody cetuximab (CmbT120C/D265C), were synthetized using GeneArt technology (Invitrogen, Regensburg, Germany). Using NheI/BssHII restriction sites for the heavy and AgeI/XbaI restriction sites for the light chain cloning, the synthesized genes were subcloned into the expression vectors pCSEH1c and pCSL3k, which harbor constant domains of a human IgG1 antibody for the eukaryotic expression of the whole heavy and light chains, respectively (14). Following vector transformation into XL1-Blue MRF’ supercompetent E. coli cells (Agilent Technologies, Waldbronn, Germany) and purification using the NucleoBond® Xtra Maxi Kit (Macherey-Nagel, Düren, Germany), the correct sequences of the heavy and light chains of CmbT120C/D265C were validated using sequencing (Microsynth Seqlab, Göttingen, Germany). Recombinant antibody expression in EXPI293F cells followed previously established protocols (14). The expressed antibody was purified using protein G affinity chromatography (Cytiva, Marlborough, MA, USA) and dialyzed against PBS. The final protein concentration of the purified antibody CmbT120C/D265C was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).

Generation of the antibody dye conjugates. The purified antibody CmbT120C/D265C was suspended in PBS with 1 mM EDTA (pH 7.4), underwent a reduction process employing 40 equimolar concentrations of Tris-(2-Carboxyethyl)phosphine-hydrochloride (TCEP, Carl Roth, Karlsruhe, Germany) for 3 h at 37°C on a shaker. Following overnight dialysis against PBS with 1 mM EDTA (pH 7.4) at 4°C, the antibody was re-oxidized utilizing 30 equimolar concentrations of dehydroascorbic acid (Sigma-Aldrich) for 4 h at room temperature. For the conjugation step, the antibody was incubated with a 10-fold equimolar concentration of WB692-CB1 at room temperature for 1 h on a shaker in the dark. To terminate the reaction, a 25-fold equimolar concentration of N-acetyl-L-cysteine (Sigma-Aldrich) was added for 15 min. Afterwards, the solution was dialyzed again overnight against PBS (pH 7.4). To eliminate unbound dye, the solution was purified using protein G affinity chromatography (Cytiva), followed by dialysis against PBS. The protein concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). TRAT120C/D265C-WB692-CB1, consisting of the recombinant cysteine-modified anti-HER antibody trastuzumab (TRAT120C/D265C) and WB692-CB1 was produced in the same way like CmbT120C/D265C-WB692-CB1 and was kindly provided by L. Klemenz.

SDS-PAGE. Validation of the antibody CmbT120C/D265C and the conjugate CmbT120C/D265C-WB692-CB1 was accomplished through SDS-PAGE under both reducing and non-reducing conditions according to manufacturer’s instructions, using Coomassie staining (Invitrogen, Carlsbad, CA, USA) and fluorescence-based imaging (λ=680 nm) (IVIS Spectrum In Vivo Imaging System; PerkinElmer, Waltham, MA, USA).

Western blot. For western blot analysis, cells were lysed in a buffer comprising 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% NaDeoxycholate, 0.05% SDS, and 1% Igepal. Protein content was quantified using the Quick Bradford Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, 100 μg of protein per lane were transferred onto nitrocellulose membranes. After blotting, EGFR and HER2 expression of the target cells were detected using anti-EGFR polyclonal rabbit IgG or anti-HER2 monoclonal rabbit antibody (both from Cell Signaling, Danvers, MA, USA). For the detection of the anti-EGFR and anti-HER2 antibodies, a horse radish peroxidase (HRP) conjugated polyclonal goat anti-rabbit antibody (Dako Denmark A/S, Glostrup, Denmark) was used. β-actin was detected as loading control using a monoclonal anti-β-actin HRP-conjugated antibody (Cell Signaling). The blotted membranes were developed utilizing an enhanced chemiluminescence (ECL) system. Protein bands were visualized and analyzed using an INTAS Chemo Star Imager (INTAS Science Imaging Instruments, Göttingen, Germany).

Flow cytometry. To evaluate the specific binding of the antibodies and conjugates to BC cells, flow cytometry was performed as described (15). A goat R-phycoerythrin-labeled anti-human IgG antibody (Southern Biotech, Birmingham, AL, USA) was used for detection. Mean fluorescence values of stained cells were quantified utilizing a FACS Calibur Flow Cytometer and the CellQuest Pro software (BD Biosciences, Heidelberg, Germany). To assess the binding affinity of the antibodies and conjugates, calculations were performed using the GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, San Diego, CA, USA). Dissociation constants (KD) were defined as the concentrations of the antibodies or conjugates that resulted in half-maximal specific binding.

Photoimmunotherapy of BC cells. For PIT, 2.5×105 cancer cells were seeded into 35 mm cell culture dishes (Thermo Fisher Scientific) and incubated for 24 h (37°C, 5% CO2). The following day, cells were treated with either medium as a control, 10 μg/ml of conjugate, 10 μg/ml of antibody, or an equimolar concentration of free dye. Twenty-four hours after treatment cells were washed with PBS, and treated with fresh medium, before getting exposed to different red light doses ranging from 32 to 128 J/cm2 (λ=690±10 nm) from a light-emitting diode (LED L690-66-60, Marubeni, Tokyo, Japan). To test repeated PIT cycles, RT112 cells were treated with 10 μg/ml CmbT120C/D265C-WB692-CB1 for 4 h, washed and exposed to a light dose of 64 J/cm2. Twenty-four hours later, cells were washed again and re-incubated with the conjugate for another 4 h and irradiated again with the same light dose. For combinatorial PIT, RT112 cells were incubated with 10 μg/ml CmbT120C/D265C-WB692-CB1 and with 10 μg/ml TRAT120C/D265C-WB692-CB1 for 24 h alone or in combination before irradiation with 64 J/cm2.

Twenty-four hours after light exposure, cells were trypsinized, stained with Erythrosine B (Logos Biosystems, Gyeonggi-do, Republic of Korea) and cell viability was analyzed using a Neubauer Counting Chamber. The number of living cells was normalized relative to the untreated control. Mean values±SD from three independent experiments were calculated using GraphPad Prism 7 software. p-Values were determined using the unpaired t-test with Welch‘s correction.

Microscopy. To analyze changes in cell morphology after PIT, cells were examined using a Zeiss AxioObserver Z.1 inverted microscope (Carl Zeiss Microscopy GmbH, Munich, Germany).

Results

For the PIT of bladder cancer cells, a conjugate consisting of a cysteine modified recombinant version of the anti-EGFR antibody cetuximab and the photosensitizer WB692-CB1 was generated. WB692-CB1 is a silicon phthalocyanine dye with a maleimide linker for coupling to SH groups, e.g., present in free cysteines, that was recently synthesized by us (Figure 1A). It has a maximum absorption wavelength of 693 nm and a maximum emission wavelength of 703 nm (Figure 1B). For the generation of the antibody dye conjugate CmbT120C/D265C-WB692-CB1 (Figure 1C), genes of the heavy chain, including cysteine mutations, as well as genes of the variable domain of the light chain (VL) of the cysteine modified cetuximab variant CmbT120C/D265C were cloned into the expression vectors pCSEH1C and pCSL3k, respectively (14). After transfection of both vectors into EXPI293F cells and purification of the cell culture supernatant using Protein G affinity chromatography, approximately 80-90 mg of pure antibody were yielded per liter cell culture. SDS-PAGE analysis under reducing conditions revealed protein bands of the expressed heavy and light chains at 50 kDa and 25 kDa, respectively, and validated the expression of the whole assembled antibody CmbT120C/D265C under non-reducing conditions with a predicted size of 150 kDa. The predicted coupling of the dye to the cysteines of the heavy chain of CmbT120C/D265C was verified by SDS-PAGE analyses under red light (λ=680 nm, Figure 1D).

Figure 1.
Figure 1.

Characterization of the anti-EGFR antibody CmbT120C/D265C and the antibody dye conjugate CmbT120C/D265C-WB692-CB1. (A) Structure of the silicon phthalocyanine dye WB692-CB1 with the maleimide linker for cysteine conjugation. (B) Absorption and emission spectra of WB692-CB1. (C) Schematic representation of the antibody dye conjugate CmbT120C/D265C-WB692-CB1 with the cysteine mutations T120C in the CH1 domain and D265C in the CH2 domain of the heavy chain for dye coupling. (D) Analysis of the antibody CmbT120C/D265C and the antibody dye conjugate CmbT120C/D265C-WB692-CB1 after separation in SDS gel under non-reducing and reducing conditions analyzed under white and red light. (E) EGFR expression in the BC target cell lines as shown by western blot. (F) Binding of the antibody CmbT120C/D265C and the antibody dye conjugate CmbT120C/D265C-WB692-CB1 to the BC cells as measured by flow cytometry.

Flow cytometric analyses were performed to assess the binding properties of the antibody dye conjugate CmbT120C/D265C-WB692-CB1 to BC cells. We used the three BC cell lines RT112, HT1376 and T24 with endogenous EGFR expression, whereas the EGFR negative CHO cell line served as control (Figure 1E). The analyses revealed high binding of the antibody CmbT120C/D265C and of the antibody dye conjugate CmbT120C/D265C-WB692-CB1 to all EGFR positive BC cells with KD values in the low nanomolar range (Figure 1F). None of the antibodies or conjugates was found to bind to CHO control cells (data not shown).

PIT of EGFR positive BC cells, using 10 μg/ml CmbT120C/D265C-WB692-CB1 and a light dose of 32 J/cm2, resulted in approximately 50% significantly reduced cell viability in all treated cell lines. No cytotoxicity was found in EGFR positive cells that were treated with uncoupled antibody or free dye. Furthermore, no cytotoxicity was induced in CmbT120C/D265C-WB692-CB1 treated cells that were not exposed to red light (Figure 2A-C). EGFR negative CHO control cells remained unaffected by any treatment (Figure 2D).

Figure 2.
Figure 2.

Photoimmunotherapy (PIT) of bladder cancer (BC) cells with the antibody dye conjugate CmbT120C/D265C-WB692-CB1. Viability of (A) RT112, (B) H1376, (C) T24 and (D) CHO cells 24 h after PIT with a light dose of 32 J/cm2. (E) Viability of CHO and RT112 cells 24 h after PIT with increased light doses. (F) Temperature of the cell culture medium during PIT with increasing light doses. (G) Morphological changes in RT112 cells 24 h after PIT with a light dose of 64 J/cm2. (H) Viability of RT112 cells 24 h after repeated PIT cycles.

To further assess the effect of light dosage on cytotoxicity, RT112 cells were treated with CmbT120C/D265C-WB692-CB1 and exposed to increasing light doses. Increasing cytotoxicity correlated with the light doses. Twenty-four hours after light exposure with 128 J/cm2, only 1.3±0.4% of the RT112 cells survived (p<0.0001, Figure 2E). Non-specific cytotoxicity through thermal cell damage as an explanation for increased cell death at higher light doses could be ruled out, as temperature increased by only about 1.5°C during irradiation with 128 J/cm2 (Figure 2F). Microscopic analyses revealed cell blebbing and debris in the EGFR-positive BC cells after PIT with the conjugate CmbT120C/D265C-WB692-CB1, whereas untreated cells did not show morphological changes (Figure 2G). To evaluate potential resistance of surviving BC cells to PIT, experiments with repeated PIT cycles, comprising incubation of the cells with 10 μg/ml conjugate for 4 h followed by irradiation with 64 J/cm2 red light, were performed. Twenty-four hours after one PIT cycle, 46.4±8.7% of the cells remained alive (p=0.0055), whereas 24 h after two PIT cycles, cell viability was reduced to 24.2±8.8% (p=0.002) (Figure 2H).

As BC is a very heterogeneous type of cancer, PIT targeting two different antigens on BC cells could be beneficial for future therapies and was hence evaluated. EGFR/HER2 positive RT112 cells (Figure 3A) were incubated with CmbT120C/D265C-WB692-CB1 and TRAT120C/D265C-WB692-CB1. Twenty-four hours after light exposure with 64 J/cm, cytotoxicity was significantly increased compared to cells that were only incubated with CmbT120C/D265C-WB692-CB1 or TRAT120C/D265C-WB692-CB1 alone. 34.0±2.0% of RT112 cells treated with CmbT120C/D265C-WB692-CB1 and 69.6±2.8% of cells treated with TRAT120C/D265C-WB692-CB1 remained alive, whereas only 18.7±3.9% of RT112 cells survived after co-incubation with both conjugates (Figure 3B, p<0.0001 and p=0.0094, respectively).

Figure 3.
Figure 3.

Photoimmunotherapy (PIT) of RT112 bladder cancer (BC) cells with the antibody dye conjugates CmbT120C/D265C-WB692-CB1 and TRAT120C/D265C-WB692-CB1. (A) EGFR and HER2 expression of the BC cell line RT112. (B) Viability of RT112 cells 24 h after PIT with single and combined incubation of CmbT120C/D265C-WB692-CB1 and TRAT120C/D265C-WB692-CB1 conjugates, respectively.

Discussion

Treatment of BC still lacks innovative therapies to efficiently and sustainably eliminate cancer cells at stages of localized disease, which results in high recurrence rates and progression risk (5). PIT is considered a promising therapeutic concept to improve the treatment of local BC, sparing healthy tissue while eliminating visible and non-visible tumor lesions. In the present study, a novel PIT approach using the newly synthesized silicon phthalocyanine dye WB692-CB1 against BC cells was established.

Our strategy to modify the antibodies with cysteines for specific dye coupling has several advantages compared to e.g., coupling to lysines, which are ubiquitously distributed in the whole antibody molecule: it enables a region-selective coupling of the dye with the possibility to regulate the dye uptake by insertion of several cysteines without affecting the antibody’s paratope and contributes to the production of homogeneous conjugate preparations (16). Moreover, mutations of amino acids in the Fc region of IgG antibodies, which interact with Fc gamma receptors or the neonatal Fc receptor (17, 18), can influence the immune responses and biodistribution of the conjugate for future use in patients in a customized manner.

Our study investigated the cytotoxic effects of PIT employing the new antibody dye conjugate CmbT120C/D265C-WB692-CB1 on BC cells expressing EGFR. Our experiments resulted in a significant and specific reduction in cell viability across all tested EGFR-positive BC cell lines. Other studies with an antibody dye conjugate consisting of the anti-EGFR antibody panitumumab and the lysine coupled phthalocyanine dye IR700 demonstrated comparable cytotoxicity at comparable light doses (19). This indicates the potential of CmbT120C/D265C-WB692-CB1 as a promising candidate for EGFR-targeted PIT in BC.

Furthermore, we found that a sufficient light dose of 128 J/cm2 effectively eliminated BC cells, meaning that there were no PIT resistant cells. In our approach, thermal effects were negligible, emphasizing the potential of prolonged light exposure times or higher light intensities for future clinical application. Additionally, repeated PIT cycles emerged as a promising strategy to enhance efficacy.

The heterogeneity of BC, characterized by genomic instability and varied receptor expressions, underscores the need for personalized and cell specific therapies (20). Therefore, we extended the focus on a combinatorial approach targeting RT112 cells expressing both EGFR and HER2 target antigens. PIT with CmbT120C/D265C-WB692-CB1 and TRAT120C/D265C-WB692-CB1 demonstrated significantly higher cytotoxicity compared to PIT with the single conjugates. Targeting multiple receptors increases the absolute number of bound and internalized antibody dye conjugates per cell, enhancing PIT efficacy. Similar findings from other studies highlight the potential of combining antibody dye conjugates for superior outcome (21, 22). In the future, molecular subtyping of patients, for example by immunohistological staining of target antigens in BC biopsies could pave the way for a personalized PIT of BC.

Conclusion

We found highly specific and light dose-dependent cytotoxicity of our new antibody dye conjugate CmbT120C/D265C-WB692-CB1 against EGFR-expressing BC cells, which makes it a promising candidate for the future PIT of BC.

Acknowledgements

The Authors thank Prof. S. Dübel, Institute of Biochemistry, Biotechnology and Bioinformatics, Technical University of Braunschweig, Germany, for providing the cloning vectors.

Footnotes

  • Authors’ Contributions

    AM, PW, RB: conceptualization; FH, IW, JS, SSS, SL, LK: data curation; FH, IW, JS, SL: formal analysis; PW: funding acquisition; FH, IW, JS, SSS, SL: investigation; IW, PW: methodology; RB, PW: project administration; AM, CG, RB, PW: resources; IW, RB, PW: supervision; FH, IW, SL, PW: validation; FH, PW: writing – original draft; FH, IW, JS, SSS, SL, LK, AM, CG, RB, PW: writing – review & editing.

  • Conflicts of Interest

    The Authors declare no conflicts of interest in relation to this study.

  • Funding

    This work was funded by the German Research Foundation (DFG, grant no. WO 2178/3-1 to PW) and by the Federal Ministry for Economic Affairs and Climate Action (BMWK, grant no. 03THW15H04 to RB and PW). This work was also supported by a grant of the Research Committee, Faculty of Medicine, University of Freiburg, Freiburg, Germany (grant no. WOL2231/23 to PW).

  • Received March 14, 2024.
  • Revision received March 27, 2024.
  • Accepted March 28, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Antibody-dye Conjugates Targeting EGFR and HER2 for the Photoimmunotherapy of Bladder Cancer
FABIAN HUBER, ISIS WOLF, JONAS STORZ, SUSANNE SCHULTZE-SEEMANN, SUSAN LAUW, LUKAS KLEMENZ, ARKADIUSZ MIERNIK, CHRISTIAN GRATZKE, REINHARD BRÜCKNER, PHILIPP WOLF
Anticancer Research May 2024, 44 (5) 1837-1844; DOI: 10.21873/anticanres.16985
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