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[Cancer Research 65, 6371-6379, July 15, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Multiepitope HER2 Targeting Enhances Photoimmunotherapy of HER2-Overexpressing Cancer Cells with Pyropheophorbide-a Immunoconjugates

Mark D. Savellano1,2, Brian W. Pogue1,2, P. Jack Hoopes1,2, Ellen S. Vitetta3 and Keith D. Paulsen1,2

1 Department of Surgery, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire; 2 Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire; and 3 Cancer Immunobiology Center, University of Texas Southwestern Medical School, Dallas, Texas

Requests for reprints: Mark D. Savellano, Surgical Research Laboratory, Dartmouth-Hitchcock Medical Center, Borwell Research Building 638E, HB# 7850, One Medical Center Drive, Lebanon, NH 03756. Phone: 603-650-5818; Fax: 603-650-4928; E-mail: mark.savellano{at}dartmouth.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Multitargeting strategies improve the efficacy of antibody and immunotoxin therapies but have not yet been thoroughly explored for HER2-based cancer treatments. We investigated multiepitope HER2 targeting to boost photosensitizer immunoconjugate uptake as a way of enhancing photoimmunotherapy. Photoimmunotherapy may allow targeted photodynamic destruction of malignancies and may also potentiate anticancer antibodies. However, one obstacle preventing its clinical use is the delivery of enough photosensitizer immunoconjugates to target cells. Anti-HER2 photosensitizer immunoconjugates were constructed from two monoclonal antibodies (mAb), HER50 and HER66, using a novel method originally developed to label photosensitizer immunoconjugates with the photosensitizer, benzoporphyrin derivative verteporfin. Photosensitizer immunoconjugates were labeled instead with a promising alternative photosensitizer, pyropheophorbide-a (PPa), which required only minor changes to the conjugation procedure. Uptake and phototoxicity experiments using human cancer cells were conducted with the photosensitizer immunoconjugates and, for comparison, with free PPa. SK-BR-3 and SK-OV-3 cells served as HER2-overexpressing target cells. MDA-MB-468 cells served as HER2-nonexpressing control cells. Photosensitizer immunoconjugates with PPa/mAb molar ratios up to ~10 specifically targeted and photodynamically killed HER2-overexpressing cells. On a per mole basis, photosensitizer immunoconjugates were less phototoxic than free PPa, but photosensitizer immunoconjugates were selective for target cells whereas free PPa was not. Multiepitope targeted photoimmunotherapy with a HER50 and HER66 photosensitizer immunoconjugate mixture was significantly more effective than single-epitope targeted photoimmunotherapy with a single anti-HER2 photosensitizer immunoconjugate, provided photosensitizer immunoconjugate binding was saturated. This study shows that multiepitope targeting enhances HER2-targeted photoimmunotherapy and maintains a high degree of specificity. Consequently, it seems that multitargeted photoimmunotherapy should also be useful against cancers that overexpress other receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The two erbB family tyrosine kinase growth factor receptors, epidermal growth factor receptor (EGFR) and HER2, have been identified as important candidates for cancer therapy. Overexpression of the EGFR in many types of cancers and of HER2 predominantly in breast, ovarian, and gastric cancers occurs frequently and is associated with aggressive disease and worse patient prognoses (1–3). Monoclonal antibodies (mAb) against these receptors have been approved for therapeutic use or are in clinical trials (2, 4). However, anti-erbB antibodies are often subcurative as single agents and are typically combined with chemotherapy or radiation to improve efficacy (2, 4–6). Targeting multiple receptors or multiple epitopes of a receptor is another way to improve the efficacy of antibody-based therapeutics (7, 8), and such multitargeting strategies have shown early promise in erbB-targeted antibody therapies (9–12).

The use of photosensitizer immunoconjugates in photodynamic therapy, termed photoimmunotherapy following the pioneering work of Mew et al. (13) in the early 1980s, is a unique approach that could further potentiate anticancer antibodies and broaden the utility of photodynamic therapy (14). Photoimmunotherapy could be especially useful for treating complex disease sites where precise targeting is necessary to prevent collateral damage of sensitive organs. Cursory preclinical animal studies and at least one preliminary clinical study present encouraging results that suggest photoimmunotherapy could be a highly effective targeted therapy (15–18). However, as with other immunotoxin therapies, the efficacy of photoimmunotherapy is limited by the amount of immunoconjugate delivered to the target cells. In this study, we investigated multitargeting strategies to boost photosensitizer immunoconjugate uptake as a way of enhancing photoimmunotherapy, which, to our knowledge, has never been rigorously examined.

Most photosensitizers used in photodynamic therapy are hydrophobic and lipophilic and tend to aggregate in aqueous solutions, so it has been cumbersome to conjugate them to antibodies. Consequently, many photosensitizer immunoconjugates used in past studies probably contained substantial amounts of free photosensitizer impurities (19–22). This problem has hindered photoimmunotherapy research and has caused a fair amount of controversy because, in many cases, target-specific effects of the photosensitizer immunoconjugates could not be clearly distinguished from nonspecific effects of free photosensitizer impurities. To eliminate uncertainties that have repeatedly arisen due to poor photosensitizer immunoconjugate quality, a novel and simple method for producing functional high-purity photosensitizer immunoconjugates was recently developed (22–24). The method has been used to make anti-EGFR photosensitizer immunoconjugates consisting of the chimeric mAb, C225 (25, 26), conjugated with a two-branched polyethylene glycol (PEG) and the photosensitizer, benzoporphyrin derivative verteporfin (BPD). PEGylated BPD-C225 photosensitizer immunoconjugates specifically targeted and photodynamically killed EGFR-overexpressing cancer cells (22–24) and also retained the receptor-blocking function of native C225 mAb.4

It was of interest to determine whether the new method for making photosensitizer immunoconjugates could be generalized to other photosensitizers and other targets. The photosensitizer pyropheophorbide-a (PPa) is an interesting alternative to BPD. In fact, a PPa derivative, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a, has been in clinical trials (27). Both PPa and BPD are promising second-generation photosensitizers, but PPa derivatives seem to induce a different phototoxic mechanism than BPD, which is likely due to differences in subcellular localization (28, 29). In terms of targets, the EGFR has been intensely studied in photoimmunotherapy and other targeted photodynamic therapy approaches (22–24, 30–33), but HER2 has received less attention (34). Consequently, further study of HER2 as a target for photoimmunotherapy is warranted. In this investigation, photosensitizer immunoconjugates were made with the alternative photosensitizer, PPa, and with two different anti-HER2 mAbs, HER50 and HER66 (11). Single-epitope targeting with a single anti-HER2 photosensitizer immunoconjugate was compared to multiepitope targeting with a mixture of the two different anti-HER2 photosensitizer immunoconjugates. We show that the PPa-labeled anti-HER2 photosensitizer immunoconjugates specifically target and photodynamically kill HER2-overexpressing cells but spare HER2-negative cells, and that multiepitope targeting can significantly enhance HER2-targeted photoimmunotherapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. The human ovarian cancer cell line SK-OV-3 and the human breast cancer cell lines SK-BR-3 and MDA-MB-468 (35–41) were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in a humidified atmosphere of 5% CO2/95% air at 37°C in DMEM/Ham's F-12 1:1, with L-glutamine (BioWhittaker Cell Culture Media, Cambrex Bio Science Walkersville, Inc., Walkersville, MD) containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin.

Antibodies. The anti-HER2 mouse mAbs, HER50 and HER66, were developed, produced, and characterized in the laboratory of Dr. Ellen S. Vitetta (11). Antibodies were prepared as stock solutions in PBS (essentially, Dulbecco's PBS solution without Ca and Mg, pH 7.4) at a concentration of ~10 mg/mL.

Preparation of photosensitizer immunoconjugates. Except for choice of photosensitizer and slight modification of the procedure used to generate the photosensitizer active ester, photosensitizer immunoconjugates were prepared as previously described (22, 24). Briefly, PPa (Frontier Scientific, Inc., Logan, UT) was converted to an active ester by reacting 3 µmol of PPa with 3 µmol of N-hydroxysuccinimide and 3 µmol of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl in 0.5 mL of methylene chloride at 4°C for ~72 hours. Crude PPa-N-hydroxysuccinimide ester was then purified by silica gel chromatography using methylene chloride as the loading solvent and ethyl acetate as the eluant. For conjugations, purified PPa-N-hydroxysuccinimide was reconstituted in DMSO at ~2.5 mmol/L concentration, as verified by absorbance measurement using a Cary 50 UV-visible spectrophotometer (Varian, Inc., Walnut Creek, CA). The extinction coefficient of PPa-N-hydroxysuccinimide in DMSO was assumed to be equal to that of PPa methyl ester in organic solvents, which is ~4.75 x 104 (mol/L)–1 cm–1 for the longest wavelength absorbance peak (29). Before PPa labeling, the mAb was PEGylated with a 10 kDa two-branched PEG-N-hydroxysuccinimide ester (Shearwater Polymers, Huntsville, AL). PPa-N-hydroxysuccinimide was then reacted with the PEGylated mAb. Crude photosensitizer immunoconjugate product was purified on Sephadex G-50 (medium particle size) spun columns. Concentrated stocks of purified photosensitizer immunoconjugates in PBS were prepared using a 50 kDa MW cutoff centrifugal filter device (Centricon YM-50, Millipore Corp., Bedford, MA).

Determining photosensitizer/monoclonal antibody molar ratios of the photosensitizer immunoconjugates. To estimate PPa content of the photosensitizer immunoconjugates, it was assumed that conjugation did not alter the extinction coefficient of PPa. This is an approximation because the absorption spectrum of conjugated PPa is clearly altered compared with that of free PPa (see Results). Photosensitizer immunoconjugate protein content was estimated by subtracting the PPa contribution to the photosensitizer immunoconjugate absorbance at 280 nm. This protein contribution to the photosensitizer immunoconjugate absorbance was then compared with the 280 nm absorbance of a standard solution of the parent mAb.

SDS-PAGE analysis of the photosensitizer immunoconjugates. Photosensitizer immunoconjugates were analyzed by SDS–PAGE (Mini-PROTEAN II electrophoresis unit, Bio-Rad Laboratories, Hercules, CA) on 5% nonreducing gels using essentially the method of Laemmli (42). Gels were imaged using a custom-built broad-beam illumination CCD camera imaging system (43). The PPa fluorescence of the gels was imaged using a 670 nm diode laser for excitation in combination with a 685 nm long-pass emission filter. After fluorescence imaging, gels were Coomassie stained and imaged under ambient room light to visualize protein content. Images were analyzed using Kodak 1D image analysis software (Eastman Kodak Company, New Haven, CT).

Cellular uptake. Cells were plated in 2 mL of media in 35-mm dishes at densities that allowed them to reach ~80% confluence after 4 days of growth. Following an initial ~48-hour period, which allowed cells to attach and begin dividing, the original media was replaced with 2 mL of fresh media containing photosensitizer immunoconjugate or free PPa. From this point onward, cells were handled under low light to prevent photosensitization. Cells were incubated with photosensitizer immunoconjugate or free PPa at 37°C, and after 40 hours, the media containing photosensitizer immunoconjugate or free PPa was removed. Dishes were then washed twice with 2 mL of PBS, and cells were removed by incubation with 250 µL lysis buffer [5 mmol/L Na2-EDTA, 10 mmol/L Tris base, 150 mmol/L NaCl, 1% Triton X-100, 0.1% protease inhibitor cocktail solution (Sigma-Aldrich, St. Louis, MO), and 1 mmol/L phenylmethanesulfonyl fluoride; protease inhibitors were added just before use] per dish for 5 minutes at room temperature, followed by the addition of 250 µL deionized distilled water per dish. The resulting 0.5 mL cell lysate samples were centrifuged at 20,000 x g for 5 minutes to remove insoluble material. A 0.4 mL aliquot of each cell lysate was mixed with 0.6 mL of 1 mol/L Tris base-1% SDS in a fluorometer cuvette to measure PPa content. Sample fluorescence was quantified by exciting at 400 nm and measuring area under the emission peak from 650 to 790 nm using a FluoroMax-3 spectrofluorometer (Jobin Yvon, Inc., Edison, NJ). Comparison to the fluorescence of free PPa standard solutions allowed absolute quantification of PPa content in the cell lysates. The remaining 0.1 mL of each cell lysate was used to quantify cell protein content by a Bradford-type assay (Bio-Rad Laboratories). To relate cell protein content to number of cells, untreated control dishes were prepared to estimate the conversion variable, number of cells per milligram of cell protein. This variable, which varies for different cell lines, was measured as follows. Control dishes were trypsinized to prepare 1 mL cell suspensions that were counted using a hemacytometer, pelleted by centrifugation at 125 x g for 7 minutes, resuspended with 1 mL PBS, and then repelleted. Cell pellets were lysed by incubation with 250 µL lysis buffer for 5 minutes at room temperature, followed by the addition of 250 µL deionized distilled water. The resulting cell lysates were centrifuged to remove insoluble material and then assayed for protein content.

Phototoxicity. Preparation of cells and incubation conditions were essentially identical to those described for cellular uptake experiments. Cells were incubated with photosensitizer immunoconjugate or free PPa at 37°C, and after 40 hours, the media containing photosensitizer immunoconjugate or free PPa was removed. Dishes were then washed once with 2 mL of PBS, and 2 mL of fresh media were added back to each dish. Cells were then immediately irradiated using a 670 nm diode laser (High Power Devices, Inc., North Brunswick, NJ). Fluence rates were ~75 to ~90 mW/cm2. After irradiation, cells were incubated 12 to 24 hours at 37°C and then assayed for viability by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (44). The MTT assay, which has frequently been used to assess chemotoxic, radiotoxic, and phototoxic sensitivities, was used in this study rather than a clonogenic assay or other alternative nonclonogenic viability assays because it is fast and relatively easy to implement.

Competition experiments. Competitions were done in both cellular uptake and phototoxicity experiments. Binding of saturating amounts of the anti-HER2 photosensitizer immunoconjugates to HER2 was competed with various concentrations of the parent mAbs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Physical characterization of the photosensitizer immunoconjugates. Photosensitizer immunoconjugates of varying PPa/mAb molar ratios were prepared from two different anti-HER2 mAbs, HER50 and HER66 (11). Absorption spectra of photosensitizer immunoconjugates with moderate loading levels (~4.5-5 PPa/mAb) and relatively high loading levels (~8-10 PPa/mAb) are shown in Fig. 1. For comparison, absorption spectra for free PPa and native HER66 mAb are also shown (the spectrum for HER50 mAb is very similar to that of HER66 mAb and is not shown). To measure spectra, photosensitizer immunoconjugates and native mAb were prepared in PBS, whereas free PPa, which is not soluble in purely aqueous solutions, was prepared in DMSO. As photosensitizer immunoconjugate loading level increases, the protein absorbance peak at ~280 nm decreases in the expected manner relative to the PPa absorbance peaks. Compared with free PPa, the photosensitizer immunoconjugates have broader PPa absorbance peaks that are blue-shifted in the Soret band region around ~400 nm and red-shifted in the Q band region around ~670 nm. Interestingly, there is a shoulder on the far-red Q band peak of the HER50 photosensitizer immunoconjugate with 8.1 PPa/mAb. This shoulder was even more prominent when the spectrum of the photosensitizer immunoconjugate was measured in 50% DMSO-50% aqueous solutions (data not shown). In general, the shoulder feature was only observed for low-purity photosensitizer immunoconjugates (>15% free PPa impurity as determined by SDS-PAGE; see below) with high loading levels (>8-10 PPa/mAb).



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  		Figure 1. Ground state absorption spectra of various photosensitizer immunoconjugates, free PPa, and native mAb. Each spectrum has been offset successively by 0.05 cm-1 absorbance units for clearer presentation. Spectra are as follows: a, HER66 photosensitizer immunoconjugate, 5.1 PPa/mAb; b, HER50 photosensitizer immunoconjugate, 4.6 PPa/mAb; c, HER66 photosensitizer immunoconjugate, 9.6 PPa/mAb; d, HER50 photosensitizer immunoconjugate, 8.1 PPa/mAb; e, free PPa, 2 µmol/L; and f, native HER66 mAb, 0.1 mg/mL. Arrow, shoulder on the far-red absorbance peak of the HER50 photosensitizer immunoconjugate with a molar ratio of 8.1 PPa/mAb.

 
The photosensitizer immunoconjugates were run on a 5% nonreducing SDS-PAGE gel alongside the parent mAbs, free PPa, and protein standards (203, 120, 90, and 52 kDa; the 52 kDa marker runs at the bromophenol blue dye front). Figure 2A shows the fluorescence image of the gel, which allows visualization of PPa content, and Fig. 2B shows the gel after Coomassie staining, which allows visualization of protein content. As we have noted elsewhere (22, 24), PEGylation of the photosensitizer immunoconjugates results in splitting of the main native mAb protein band running at ~160 kDa into a ladder of three to four major photosensitizer immunoconjugate bands of equal or greater molecular weight. These major photosensitizer immunoconjugate bands correspond to zero, one, two, and three PEG molecules per photosensitizer immunoconjugate. Photosensitizer immunoconjugate bands corresponding to ≥4 PEG molecules per photosensitizer immunoconjugate as well as heavy molecular weight photosensitizer immunoconjugate aggregates are observed, but these seem to be minor species. In addition, low molecular weight protein impurities in the mAbs and their analogous conjugate impurities in the photosensitizer immunoconjugates are observed, but these also seem to be minor species.



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  		Figure 2. Five percent nonreducing SDS-PAGE gel of various photosensitizer immunoconjugates and controls. A, fluorescence image of gel before Coomassie staining. B, white light illumination image of Coomassie-stained gel. Arrows, level of the bromophenol blue dye front. Lane 1, protein standards; lane 2, native HER50 mAb; lane 3, HER50 photosensitizer immunoconjugate, 4.6 PPa/mAb; lane 4, HER50 photosensitizer immunoconjugate, 8.1 PPa/mAb; lane 5, native HER66 mAb; lane 6, HER66 photosensitizer immunoconjugate, 5.1 PPa/mAb; lane 7, HER66 photosensitizer immunoconjugate, 9.6 PPa/mAb; lane 8, empty; lane 9, free PPa.

 
Image analysis of Fig. 2A allows assessment of the amount of free PPa impurity in the various photosensitizer immunoconjugate preparations based on the fact that conjugated PPa runs at or above the bromophenol blue dye front whereas unconjugated free PPa runs below it. Assuming conjugated PPa has the same fluorescence quantum yield as free PPa in the highly denaturing SDS-PAGE environment, we measured 2.3% and 20.9% free PPa impurity for the HER50 photosensitizer immunoconjugates with 4.6 and 8.1 PPa/mAb, respectively, and 2.4% and 10.8% free PPa impurity for the HER66 photosensitizer immunoconjugates with 5.1 and 9.6 PPa/mAb, respectively. Because conjugation can decrease fluorescence quantum yields (22, 24), these impurity measurements should be regarded as upperbound estimates. Only photosensitizer immunoconjugates with roughly ≤10% free PPa impurity were used in the cellular uptake and phototoxicity experiments.

Free pyropheophorbide-a phototoxicity experiments. Free PPa is a potent photosensitizer (28, 29) but has no inherent targeting specificity. To show that free PPa is nonspecific and to establish a baseline for gauging the performance of the photosensitizer immunoconjugates, free PPa phototoxicity experiments were conducted with various cancer cell lines. Incubations were for 40 hours at 37°C so that conditions would be comparable to those of the photosensitizer immunoconjugate experiments, for which prolonged incubations were previously deemed necessary to achieve appreciable cell killing (24, 33). Figure 3A to C shows phototoxicity data for a range of free PPa concentrations and light doses. Although some variation in photosensitivity among the cell lines was observed, the data confirm that free PPa is generally nonspecific. Greater than 90% cell killing (LD90) was easily achieved for all cell lines using only moderate doses of free PPa and light. At L/L free PPa, LD90 light doses for SK-BR-3, SK-OV-3, and MDA-MB-468 cells were roughly 8, 9, and 2 J/cm2, respectively.



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  		Figure 3. Free PPa phototoxicity and uptake experiments. Cells were incubated with free PPa at the indicated concentrations for 40 hours at 37°C and then irradiated with a range of light doses for phototoxicity experiments or prepared as lysates for cellular uptake measurements. A to C, phototoxicity experiments with SK-BR-3, SK-OV-3, and MDA-MB-468 cells, respectively. D, uptake experiments with the various cell lines. Bars, SDs for data that were collected at least in triplicate.

 
Free pyropheophorbide-a cellular uptake experiments. Free PPa cellular uptakes for incubation conditions identical to those of the phototoxicity experiments are shown in Fig. 3D. Uptakes increased as the incubation concentration of free PPa was increased. However, uptakes were negligible at 50 nmol/L, suggesting that free PPa binds strongly to serum proteins in the media, which effectively reduces its cellular availability at concentrations ≤50 nmol/L. Because phototoxic dose is directly proportional to photosensitizer uptake (33), these data explain why all cell lines exhibited only a mild response to photodynamic therapy treatment at 50 nmol/L free PPa and became significantly more photosensitive at concentrations above 50 nmol/L free PPa. Likewise, the fact that SK-OV-3 cells took up less free PPa than SK-BR-3 and MDA-MB-468 cells seems to explain why SK-OV-3 cells were somewhat more resistant to photodynamic therapy treatment with free PPa.

HER2-targeted photoimmunotherapy experiments. The two HER2-overexpressing cell lines, SK-BR-3 and SK-OV-3, express ~1 x 106 to 2 x 106 and ~0.5 x 106 to 1 x 106 HER2 receptors per cell, respectively (37, 38, 45). Nontarget MDA-MB-468 cells do not express detectable levels of HER2 (38, 46) but do overexpress the related erbB family growth factor receptor EGFR at ~0.3 x 106 to 2 x 106 receptors per cell (30, 41, 47). Cells were targeted with photosensitizer immunoconjugates constructed from HER50 and HER66 mAbs. Cross-blocking studies have established that these mAbs bind to two different epitopes on the extracellular domain of HER2 (11). For single-epitope targeting, cells were incubated either with HER50 photosensitizer immunoconjugate or with HER66 photosensitizer immunoconjugate. For multiepitope targeting, cells were incubated with a 1:1 mixture of HER50 and HER66 photosensitizer immunoconjugates. Incubations were for 40 hours at 37°C.

Representative photoimmunotherapy experiments are shown in Fig. 4A to D. As expected, cell killing induced by HER2-targeted photoimmunotherapy correlated with HER2 expression levels. SK-BR-3 and SK-OV-3 cells were susceptible to various HER2-targeted photoimmunotherapy regimens (Fig. 4A-C), whereas nontarget MDA-MB-468 cells were largely unaffected (Fig. 4D). Moreover, SK-BR-3 cells were more responsive than SK-OV-3 cells to both single-epitope and multiepitope targeted photoimmunotherapy, reflecting the fact that SK-BR-3 cells express about twice more HER2 receptors per cell than SK-OV-3 cells.



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  		Figure 4. HER2-targeted photoimmunotherapy experiments. Cells were incubated with HER66 photosensitizer immunoconjugate and/or HER50 photosensitizer immunoconjugate for 40 hours at 37°C and then irradiated with a range of light doses. PPa/mAb molar ratios of the various photosensitizer immunoconjugates are indicated in the key. Phototoxicity experiments were conducted with HER2-overexpressing SK-BR-3 cells (A and B), HER2-overexpressing SK-OV-3 cells (C), and HER2-negative MDA-MB-468 cells (D). For single-epitope targeting experiments using only one photosensitizer immunoconjugate, incubation concentrations were 150 nmol/L PPa content (A, C, and D) and 300 nmol/L PPa content (B). For multi-epitope targeting experiments using both HER66 and HER50 photosensitizer immunoconjugates, incubation concentrations were 75 nmol/L PPa content of HER66 photosensitizer immunoconjugate + 75 nmol/L PPa content of HER50 photosensitizer immunoconjugate (A) and 150 nmol/L PPa content of HER66 photosensitizer immunoconjugate + 150 nmol/L PPa content of HER50 photosensitizer immunoconjugate (B, C, and D). Bars, SDs for data that were collected at least in triplicate.

 
When the total concentration of a 1:1 mixture of moderately labeled HER50 and HER66 photosensitizer immunoconjugates with 4.6 to 5.1 PPa/mAb was only 150 nmol/L PPa content (i.e., individual photosensitizer immunoconjugate concentrations of 75 nmol/L PPa content), multiepitope targeted photoimmunotherapy showed no remarkable enhancement over single-epitope targeted photoimmunotherapy with a single moderately labeled anti-HER2 photosensitizer immunoconjugate at 150 nmol/L PPa content (Fig. 4A). However, when the total concentration of a 1:1 mixture of moderately labeled HER50 and HER66 photosensitizer immunoconjugates was at least 300 nmol/L PPa content (i.e., individual photosensitizer immunoconjugate concentrations of at least 150 nmol/L PPa content), multiepitope targeted photoimmunotherapy significantly enhanced killing of the HER2-overexpressing cells in comparison to single-epitope targeted photoimmunotherapy (Fig. 4B and C).

As an alternative approach, we examined whether single-epitope targeting using a single photosensitizer immunoconjugate with a relatively high PPa/mAb molar ratio could produce a photoimmunotherapy response comparable to that of multiepitope targeting using a 1:1 mixture of moderately labeled HER50 and HER66 photosensitizer immunoconjugates with 4.6 to 5.1 PPa/mAb. We made a HER66 photosensitizer immunoconjugate of decent purity with a high labeling ratio of 9.6 PPa/mAb, which is nearly equal to the sum of the respective PPa/mAb ratios of the moderately labeled HER50 and HER 66 photosensitizer immunoconjugates. As seen in Fig. 4A and B, for SK-BR-3 cells, the phototoxic response to single-epitope targeted photoimmunotherapy using 150 nmol/L PPa content of HER66 photosensitizer immunoconjugate with 9.6 PPa/mAb is indeed roughly comparable to that of multiepitope targeted photoimmunotherapy using 300 nmol/L PPa content of a 1:1 mixture of the moderately labeled HER50 and HER66 photosensitizer immunoconjugates.

Photosensitizer immunoconjugate binding specificity experiments. HER2-overexpressing SK-BR-3 cells and HER2-negative MDA-MB-468 cells were incubated with 150 nmol/L PPa content of anti-HER2 photosensitizer immunoconjugate with or without competing antibody for 40 hours at 37°C. Figure 5A shows that SK-BR-3 uptake of HER66 photosensitizer immunoconjugate was progressively inhibited when competed with increasing amounts of HER66 mAb but was only slightly inhibited when competed with HER50 mAb. Similarly, Fig. 5B shows that SK-BR-3 uptake of HER50 photosensitizer immunoconjugate was progressively inhibited when competed with increasing amounts of HER50 mAb but was only slightly inhibited when competed with HER66 mAb. In experiments with HER2-negative MDA-MB-468 cells, noncompeted HER66 and HER50 photosensitizer immunoconjugate uptakes were (0.72 ± 0.16) x 10–3 and (1.12 ± 0.48) x 10–3 fmol of PPa per cell, respectively, or, equivalently, ~26.8 and ~13.2 times less than the corresponding noncompeted photosensitizer immunoconjugate uptakes for SK-BR-3 cells. Overall, these results show that the HER66 and HER50 photosensitizer immunoconjugates specifically target HER2 and do not significantly cross-block each other.



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  		Figure 5. Photosensitizer immunoconjugate competitive uptake experiments. HER2-overexpressing SK-BR-3 cells were incubated for 40 hours at 37°C with 150 nmol/L PPa content of HER66 photosensitizer immunoconjugate (A) or HER50 photosensitizer immunoconjugate (B) plus the indicated amount of competing mAb, and were then prepared as lysates to measure cellular uptakes. Data were collected in triplicate; bars, SD.

 
As a corollary to the competitive uptake experiments, competitive phototoxicity experiments were also conducted under identical incubation conditions. When SK-BR-3 cells were treated with HER66 photosensitizer immunoconjugate (9.6 PPa/mAb), a 40 J/cm2 light dose reduced cell viability by ~95% compared with unirradiated cells. However, when SK-BR-3 cells were treated with HER66 photosensitizer immunoconjugate and a 4-fold excess of HER66 mAb, a 40 J/cm2 light dose reduced cell viability by only ~4% compared with unirradiated cells. Similarly, when SK-BR-3 cells were treated with HER50 photosensitizer immunoconjugate (5.7 PPa/mAb), a 40 J/cm2 light dose reduced cell viability by ~84% compared with unirradiated cells, but when cells were treated with HER50 photosensitizer immunoconjugate and a 4-fold excess of HER50 mAb, a 40 J/cm2 light dose reduced cell viability by only ~3% compared with unirradiated cells. These data are consistent with the competitive uptake results in Fig. 5 and further show that the phototoxic effects of the anti-HER2 photosensitizer immunoconjugates are mediated by specific HER2 targeting.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PPa is chemically similar to BPD, and BPD analogues have even been synthesized from PPa (48, 49). Consequently, the procedure to make PPa-labeled photosensitizer immunoconjugates was much the same as that originally devised to make BPD-labeled photosensitizer immunoconjugates (22, 24), except that the method for generating the photosensitizer-N-hydroxysuccinimide active ester was slightly modified. By increasing the stoichiometric ratio of PPa-N-hydroxysuccinimide to mAb, PPa/mAb molar ratios of the photosensitizer immunoconjugates could be increased, but the free PPa impurity in the photosensitizer immunoconjugates also increased. Efforts to completely remove residual free PPa impurity from the photosensitizer immunoconjugates were unsuccessful, indicating that some PPa did not covalently attach to the mAb and instead became tightly bound via noncovalent interactions. In general, the HER66 mAb could be covalently labeled to somewhat higher PPa/mAb molar ratios than the HER50 mAb, although both mAbs are of the immunoglobulin G1k isotype (11). To keep free PPa impurity roughly ≤10%, the PPa/mAb molar ratio of the HER66 photosensitizer immunoconjugate could not exceed ~10, and the PPa/mAb molar ratio of the HER50 photosensitizer immunoconjugate could not exceed ~7.5. The different photosensitizer loading capacities of different mAbs are probably due to several factors. One factor is the number of mAb primary amines available for covalent linkage of photosensitizer. A subtler factor is the number of mAb hydrophobic sites (i.e., hydrophobic pockets that are either inherent to the mAb structure or created due to covalent attachment of photosensitizer) because noncovalent binding of photosensitizer to the mAb via hydrophobic interactions competes with covalent linkage of photosensitizer to the mAb via amide bond formation (22).

If target specificity is to be maintained, the free photosensitizer impurity of a photosensitizer immunoconjugate must be limited because any noncovalently bound free photosensitizer impurity can dissociate from the photosensitizer immunoconjugate upon exposure to serum proteins. In this investigation, only photosensitizer immunoconjugates containing roughly ≤10% free PPa impurity as determined by SDS-PAGE (Fig. 2) were used in cell studies, and photosensitizer immunoconjugate incubation concentrations never exceeded 300 nmol/L PPa content. Consequently, in cell studies with the photosensitizer immunoconjugates, free PPa impurity was always less than ~30 nmol/L, which was expected to have a negligible effect based on experiments with free PPa (Fig. 3). Indeed, our data (Figs. 4 and 5) clearly show that the phototoxic effects of the anti-HER2 photosensitizer immunoconjugates are mediated via specific HER2 targeting and are not due to the nonspecific effects of free photosensitizer impurity in the photosensitizer immunoconjugates.

Both HER50 and HER66 photosensitizer immunoconjugates had lower binding affinities than their respective parent mAbs. This was expected because conjugation can sterically hinder or disrupt mAb antigen binding sites. Conjugation seemed to impair HER50 binding to a greater extent than it impaired HER66 binding. For example, HER66 photosensitizer immunoconjugate (~9.6 PPa/mAb) uptake was inhibited by 0.25-fold HER66 mAb to about half of its noncompeted uptake, whereas HER50 photosensitizer immunoconjugate (~5.7 PPa/mAb) uptake was inhibited by 0.25-fold HER50 mAb to roughly a third of its noncompeted uptake (Fig. 5). Moreover, in single-epitope targeting experiments, increasing the photosensitizer immunoconjugate concentration from 150 to 300 nmol/L PPa content resulted in only a mild increase in HER66 photosensitizer immunoconjugate phototoxicity but markedly increased HER50 photosensitizer immunoconjugate phototoxicity (Fig. 4A and B).

The effects of photosensitizer immunoconjugate binding affinity were also apparent in multiepitope targeting experiments. At 150 nmol/L PPa content, multiepitope targeted photoimmunotherapy with a 1:1 mixture of HER50 and HER66 photosensitizer immunoconjugates was not any better than single-epitope targeted photoimmunotherapy with a single photosensitizer immunoconjugate (Fig. 4A). However, at 300 nmol/L PPa content, multiepitope targeted photoimmunotherapy with the 1:1 mixture of HER50 and HER66 photosensitizer immunoconjugates was substantially more effective than single-epitope targeted photoimmunotherapy (Fig. 4B and C). Therefore, multiepitope targeting can provide a distinct advantage over single-epitope targeting, but photosensitizer immunoconjugate binding must approach saturation to achieve maximal efficacy.

In addition to multiepitope targeting, another way to enhance photoimmunotherapy is to increase the photosensitizer/mAb molar ratios of the photosensitizer immunoconjugates. We found that single-epitope targeting using 150 nmol/L PPa content of a highly labeled HER66 photosensitizer immunoconjugate with 9.6 PPa/mAb (Fig. 4A) was as phototoxic as multiepitope targeting using 300 nmol/L PPa content of a 1:1 mixture of moderately labeled HER50 and HER66 photosensitizer immunoconjugates with 4.6 to 5.1 PPa/mAb (Fig. 4B). These results seem to suggest that multiepitope targeting provides no benefit over single-epitope targeting, but it must be kept in mind that photosensitizer/mAb ratios do not have to be as high in multiepitope targeted photoimmunotherapy to achieve the same phototoxic response as single-epitope targeted photoimmunotherapy. Because lower photosensitizer/mAb ratios generally permit better preservation of photosensitizer immunoconjugate binding affinity, multiepitope targeting with a mixture of moderately labeled photosensitizer immunoconjugates has an advantage over single-epitope targeting with a single highly labeled photosensitizer immunoconjugate. Moreover, free photosensitizer impurity was less for multiepitope targeting because the HER66 photosensitizer immunoconjugate with 9.6 PPa/mAb contained ~10% free PPa impurity, whereas the HER50 and HER66 photosensitizer immunoconjugates with 4.6 to 5.1 PPa/mAb contained only ~2.4% free PPa impurity. As a further demonstration of the superiority of multiepitope targeting, we have subsequently conducted photoimmunotherapy experiments using 300 nmol/L PPa content of a 1:1 mixture of HER50 and HER66 photosensitizer immunoconjugates with moderately high labeling ratios (6.8-8.0 PPa/mAb). With this photosensitizer immunoconjugate mixture, SK-BR-3 cell viability was reduced to 12.9%, 5.8%, and 3.6% relative to untreated cells for light doses of 10, 20, and 40 J/cm2, respectively, which is a markedly stronger phototoxic response than that observed for single-epitope targeted photoimmunotherapy using the HER66 photosensitizer immunoconjugate with 9.6 PPa/mAb (Fig. 4A).

Taken as a whole, our data show that multiepitope targeting and the maximization of photosensitizer/mAb ratios are both effective strategies for amplifying photosensitizer immunoconjugate cellular uptake and, in turn, for enhancing photoimmunotherapy efficacy. Even so, when photosensitizer immunoconjugate uptakes reached levels comparable to free PPa uptakes, photosensitizer immunoconjugates still were not as photodynamically potent as free PPa. For example, at 150 nmol/L PPa content, SK-BR-3 cellular uptakes for free PPa (Fig. 3D) and for the HER66 photosensitizer immunoconjugate with 9.6 PPa/mAb (Fig. 5A) were roughly equal (~0.02 fmol of PPa per cell, or, equivalently, ~1.2 x 107 PPa molecules per cell), but in corresponding phototoxicity experiments, the LD90 light doses were ~8 J/cm2 for free PPa (Fig. 3A) and ~20 J/cm2 for the HER66 photosensitizer immunoconjugate (Fig. 4A). However, in view of the high specificity of the photosensitizer immunoconjugates, these results also indicate that it is possible to at least partially compensate for the lower phototoxicity of the photosensitizer immunoconjugates by using higher light doses.

The lower phototoxicity of the anti-HER2 photosensitizer immunoconjugates compared with free PPa mirrors results from earlier studies with a BPD-labeled photosensitizer immunoconjugate (22, 24). On a per mole basis, the BPD-labeled photosensitizer immunoconjugate was less photodynamically potent than free BPD due to changes in the photophysical and photochemical properties of the photosensitizer as well as modification of the photosensitizer subcellular localization pattern (33). As noted earlier, the dye portion of the absorption spectra for the anti-HER2 photosensitizer immunoconjugates is strikingly different from that of free PPa (Fig. 1). This suggests that the anti-HER2 photosensitizer immunoconjugates are quenched due to static concentration effects (24, 33) and, as a consequence, are less photochemically active than free PPa in cellular environments. With respect to subcellular localization, it is well documented that cells internalize anti-erbB mAb conjugates via receptor-mediated endocytosis and then route them to the lysosomes for degradation (22, 50).4 In contrast, small hydrophobic/lipophilic photosensitizer molecules, such as free PPa or free BPD, rapidly sequester in the hydrophobic compartments of cellular organelles, including mitochondria (29, 51). Because mitochondria have been implicated in the rapid induction of apoptosis following photodynamic therapy (28, 52) and seem to be a more critical target than the lysosomes, subcellular localization is likely another important reason why the photosensitizer immunoconjugates are less phototoxic than the free photosensitizer.

The possibility also exists that small subpopulations of cells in the HER2-overexpressing cell lines may not express enough HER2 to be effectively targeted. Although we did not measure the distribution of HER2 cellular expression levels for the cell lines in this study, Spiridon et al. (11) showed by fluorescence-activated cell sorting analysis that ~4% to ~8% of HER2-overexpessing BT-474 cells did not stain positive with the HER50 and HER66 mAbs. Therefore, although multiepitope targeting and maximization of the photosensitizer/mAb ratios of the photosensitizer immunoconjugates enabled high average uptakes of ≥1 x 107 PPa molecules per target cell, which is well within the photosensitizer dosage range required for potent photodynamic killing (33), a small subpopulation of target cells with relatively low HER2 levels may not have taken up enough anti-HER2 photosensitizer immunoconjugates to be killed by photoimmunotherapy.

In summary, we have shown that multiepitope targeting can significantly enhance the efficacy of HER2-targeted photoimmunotherapy. More generally, our results indicate that multiepitope targeting should be advantageous for photoimmunotherapy of other cell-surface receptors that are frequently overexpressed in various cancers (e.g., the EGFR, Met, and fibroblast growth factor receptors). Although it is clearly feasible to exploit multitargeting in photoimmunotherapy and still maintain a high degree of specificity, a shortcoming of photoimmunotherapy continues to be the reduced phototoxicity of the photosensitizer immunoconjugate in comparison to the free photosensitizer. Surmounting this problem may require further refinements in photosensitizer immunoconjugate design, which might include incorporating cleavable linkers (53) to allow photosensitizer release from the mAb once the photosensitizer immunoconjugate has reached its target (this could increase photosensitizer immunoconjugate potency by reducing adverse static concentration quenching effects and by liberating the photosensitizer from the lysosomes so that it can sequester in other subcellular sites that are more photodynamically sensitive; ref. 33). Nevertheless, our current photosensitizer immunoconjugate constructs have proven highly effective in vitro. The next step is in vivo experimentation with the photosensitizer immunoconjugates, and we are now pursuing such studies in tumor xenograft models.


   Acknowledgments
 
Grant support: NIH grants R01CA69544, P01CA84203, and P30CA23108.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Robert Gilmont, Riley Rees, and Stephen Rand who provided the initial inspiration for this research. Thanks are also due to Dr. Tayyaba Hasan's group who helped sustain the vision for this work and provided much of the early support that has opened many avenues for future inquiry.


   Footnotes
 
4 A.C.E. Moor, K.W. Mulder, M.D. Savellano, W. Yu, P.K. Selbo, T. Hasan. Photoimmunotargeting of the epidermal growth factor receptor in vitro. Int J Cancer, submitted for publication. Back

Received 2/ 8/05. Revised 5/ 2/05. Accepted 5/ 3/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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