Novel method for in vitro depletion of T cells by monoclonal antibody-targeted photosensitization

Journal of Immunological Methods 211 Ž1998. 139–146

Novel method for in vitro depletion of T cells by monoclonal antibody-targeted photosensitization Timea Berki ) , Peter ´ Nemeth ´ Department of Immunology and Biotechnology, UniÕersity Medical School of Pecs, ´ POB 99, Pecs ´ H-7643, Hungary Received 24 June 1997; revised 17 November 1997; accepted 18 November 1997

Abstract An immunotargeting method Žcalled photo-immunotargeting. has been developed for selective in vitro cell destruction. The procedure combines the photosensitizing Žtoxic. effect of light-induced dye-molecules, e.g., hematoporphyrin ŽHP. and the selective binding ability of monoclonal antibodies ŽmAb. to cell surface molecules. The photosensitizer HP molecules were covalently attached to monoclonal antibodies Ža-Thy-1. recognizing an antigen on the surface of T lymphocytes, and used for T cell destruction. To increase the selectivity of the conventional targeting methods, a physical activation step Žlocal light irradiation. as a second degree of specificity was employed. The HP in conjugated form was sufficient to induce T cell Žthymocytes, EL-4 cell line. death after irradiation at 400 nm, at tenfold lower concentration compared to the photosensitizing effect of unbound HP. The selective killing of T lymphocytes Žbearing the Thy-1 antigen. in a mixed cell population was demonstrated after a treatment with the phototoxic conjugate and light irradiation. This method can be useful for selective destruction of one population Žtarget cell. in an in vitro heterogeneous cell mixture, e.g., in bone marrow transplants for T cell depletion to avoid graft vs. host reaction. q 1998 Elsevier Science B.V. Keywords: Immunotargeting; Photosensitization; Monoclonal antibody conjugate; T lymphocyte; Thy-1

1. Introduction The selective action of different drug molecules on the target cells is the term of reduction side effects of pharmaceuticals used in cancer therapy or in vitro cytotoxic methods. To develop new highly selective cytotoxic agents is a great effort, since no Abbreviations: HP, Hematoporphyrin; mAb, Monoclonal antibody; PBS, Phosphate buffered saline; HPD, Hematoporphyrin derivative; TLC, Thin-layer chromatography ) Corresponding author. Tel.: q36-72-324-122rext. 1994; fax: q36-72-324-122rext. 1993; e-mail: [email protected].

perfect drug without side-effects has been created yet. Recent progress in this area has relied upon the use of carrier molecules Žhormones, lectins, antibodies. that bind to the surface components of the selected cells ŽDevanathan et al., 1990. and guide the toxic molecule Žchemicals, plant or bacterial toxins. to the target. Such drug-carrier conjugates have been used in vivo in tumor therapy ŽKing et al., 1994. and in vitro in cancer research ŽOseroff et al., 1987., as well as in bone marrow transplantation ŽKaneko et al., 1994.. The success of the in vivo and in vitro application of these conjugates was limited by the highly toxic drug molecules killing other cell types

0022-1759r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 7 . 0 0 2 0 1 - 9

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T. Berki, P. Nemethr Journal of Immunological Methods 211 (1998) 139–146 ´

labeled non-specifically by the conjugate ŽVitetta and Thorpe, 1987., or by the altered antigen expression of tumor cells ŽTagliaferri et al., 1994.. Our aim was to decrease the toxicity and to increase the selectivity of the targeting methods by combining a physical activation step as a second means of specificity. Photosensitizing drugs Žpsoralens, porphyrins, etc.. activated by visible light are short-lived cytotoxic molecules used in the photodynamic therapy ŽDougherty and Marcus, 1992; Statius van Eps et al., 1997. and diagnosis of tumors ŽLipson et al., 1961.. Accumulation of photosensitizer molecules in the tumor cells ŽKessel and Chou, 1983; Veenhuizen et al., 1996. and their activation by light leads to the formation of their toxic triplet state that initiates a free-radical chain-reaction ŽPoto ´ ´ and Berki, 1989. and provide other possibilities for the selective cell destruction ŽPenning and Dubbelman, 1994.. However, this method has also a variety of side-effects, mainly caused by the retained phototoxic molecules in normal tissues that are exposed to visible light, e.g., in the skin or eye ŽHe et al., 1989; Hawkins et al., 1986.. Combining the light-induced photosensitization potential of the above drugs with targeting methods, a new type of selective cytotoxic procedure, called photo-immunotargeting, has been developed ŽOseroff et al., 1987; Pogrebniak et al., 1993.. This system consists of photosensitizer dye molecule coupled to carrier molecule ŽmAb. which can be converted into cytotoxic agent only if activated by a visible light source. In this study, we used a monoclonal antibody ŽBalogh et al., 1992. specific for a T cell surface antigen Ža-Thy-1. that was covalently bound to hematoporphyrin ŽHP. molecules. The selective binding of the conjugate to mouse thymocytes and mouse T cell lines was demonstrated. The biological effects of activating the conjugate at 400 nm wavelength peak were investigated. The selectivity of the method was evaluated by treating a mixed cell population of B and T cell lines with the HP-a-Thy-1 conjugate with immunological Žflow cytometric. identification of the survivor cells. The model may present an alternative in different medical applications, e.g., in bone marrow transplantation to remove inappropriate T cells, or for tumor targeting as well.

2. Materials and methods 2.1. Chemicals Hematoporphyrin dihydrochloride, 1-ethyl-3,3-dimethylaminopropyl-carbodiimide-HCl ŽEDCl., and all the tissue culture reagents were obtained from Sigma Chemical, St. Louis, MO, USA. The hematoporphyrin derivative was prepared according to the method of Lipson et al. Ž1961.. 2.2. Cells, cell lines The Thy-1 positive EL-4 cell line Žmouse T cell lymphoma, ATCC TIB 39, maintained in the repository of our Laboratory. and mouse thymocytes were used to demonstrate the selective binding ability of the a-Thy-1 mAb and its conjugate. Thymocytes were isolated from 4-week-old BALBrc mice ŽCharles Rivers Laboratories. and adjusted to the concentration of 2 = 10 6rml in PBS for in vitro studies. For control experiments, the following Thy-1 negative cell lines were used: A20 Žmouse B cell lymphoma ATCC TIB 208., Sp-2r0-Ag14 mouse myeloma cell line ŽATCC CRL 1581. and a mouse hybridoma cell line secreting IgG1 monoclonal antibody against insulin Ža-Insulin mAb, Clone No. E2E3.. The rat–mouse hybridoma cell line ŽIBL-1. secreting antibody ŽIgG1. against the Thy-1 antigen Ža-Thy-1. was produced in our department ŽBalogh et al., 1992.. 2.3. Production and purification of the a-Thy-1 monoclonal antibody The IBL-1 hybridoma cell line was cultured in vitro in DMEM containing 10% fetal calf serum ŽFCS low IgG; Gibco.. The monoclonal antibody was purified from hybridoma supernatant with FPLC using Protein-G affinity column ŽPharmacia LKB, Sweden. equilibrated with 0.02 M phosphate buffer, pH 7.2 containing 0.1% sodium azide. The elution was carried out with 0.1 M glycine pH 2.5, and the collected fractions were neutralized with 1 M Tris base. The antibody-containing samples were dialyzed overnight against 0.01 M phosphate buffer pH 7.2.

T. Berki, P. Nemethr Journal of Immunological Methods 211 (1998) 139–146 ´

2.4. Preparation and characterization of monoclonal antibody-hematoporphyrin conjugate Conjugation was carried out with a slight modification of the original method of Mew et al. Ž1983.. Briefly, 2 mg of hematoporphyrin–dihydrochloride dissolved in the mixture of 150 m l H 2 O and 100 m l N,-N-dimethylformamide was added to 2 mg of 1ethyl-3-Ž3-dimethyl-aminopropyl.-carbodiimide-HCl dissolved in 100 m l H 2 O. After 30 min, this solution was mixed for 5 h with 2 mg mAb in 0.01 M phosphate buffer pH 7.2. The pH was maintained between 6.0 and 7.0 during this period, and the procedure was carried out in the dark. The reaction was terminated by adding 20 m l monoethanolamine to the mixture. The solution was dialyzed against 0.001 M, then 0.01 M phosphate buffer and finally against PBS, pH 7.2 at 48C for 48 h in the dark, then freeze-dried, re-dissolved in dd H 2 O and immediately passed through a Sephadex G-25 column, equilibrated with PBS. The colored conjugate was eluted, while the unconjugated, free HP molecules remained tightly bound Žred. to the column Žcould not be removed with PBS or by treatment with BSA or urea.. The purity of the conjugate was tested by thin-layer chromatography ŽTLC. ŽBelcher et al., 1970.. The conjugate was stabilized with 0.1% BSA and was filtered through a 0.2 m m Millipore filter. The amount of HP bound was determined spectrophotometrically by measuring and comparing the absorbance of the conjugate and of HP samples at known concentration at 280 and 405 nm. The protein–HP ratio was calculated as 0.136. The binding ability of the free antibody and the conjugate was measured by indirect immunofluorescence method and by flow cytometry on different cell lines. Bleaching of the weak, direct fluorescence of HP molecules from the conjugate bound to the cell surface made its direct immunofluorescence detection impossible. 2.5. Flow cytometry For the demonstration of selective binding ability of the a-Thy-1 antibody and its HP-conjugate to different cell lines, an indirect labeling was used. Briefly, the target cell populations were incubated with the first antibody or its HP conjugate at appro-

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priate concentration in PBS Žcontaining 10% FCS and 0.1% azide. at 48C for 30 min in the dark, washed 3 times with PBSrazide and incubated for further 30 min at 48C with FITC-labeled sheep-antirat IgG. After washing with PBSrazide, the cells were fixed with 1% buffered formaldehyde and stored at 48C until analysis. To determine the composition of the photosensitized cell mixtures ŽEL-4 and Sp-2r0 or hybridoma at the ratio of 1:1 at the beginning of the experiment. 24 h after the irradiation, a double labeling method was performed. The cells were incubated firstly with an anti-CD24 monoclonal antibody ŽIBL-3r14 mAb, produced in our department. for B cell staining, followed with a sheep-anti-rat Ig-FITC antibody. After a blocking step with normal rat serum, a biotin-labeled anti-Thy-1 monoclonal antibody was added. After three washing steps, the streptavidin– Phycoerythrin ŽPE. conjugate Ždiluted in PBS-10% FCS, 0.1% azide. was added. After washing with PBSrazide, the cells were fixed with 1% buffered formaldehyde and stored at 48C until analysis using a Becton Dickinson FACSCalibur and Cell Quest software. 2.6. In Õitro photosensitization assays Different cell types and cell mixtures were incubated with HPD, HP-a-Thy-1 conjugate and a-Thy-1 mAb at different concentrations Ž0.1–1.5 m grml HP content. in DMEM-10% FCS for 1 h at 48C in the dark. After three washing steps, 5 = 10 5 cells per well in 96 well microplates ŽU-shape, Greiner, Germany. were irradiated with a Hg-lamp Žwith U-205 and G-241 filtered light. at 400 nm wavelength peak Ž2–12 Jrcm2 .. The cells were returned into the CO 2 incubator Ž5% CO 2 content, 378C, humidified atmosphere. and cultured and examined during a 48-h period. The morphological changes of the irradiated samples were examined by inverted microscopy. The cell viability of 10 m l samples from each well of the cell culture was determined by trypan blue dye exclusion test ŽTennant, 1964. and by the colorimetric cell proliferation enzyme assay of Landegren Ž1984.. 2.7. Colorimetric cell proliferation assay p-Nitrophenol, N-acetyl-b-D-glucosaminide at 7.5 mM was dissolved in 0.1 M citrate buffer, pH 5.0

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and mixed with equal volume of 0.5% Triton X-100 in water. The hexosaminidase substrate was aliquoted and stored at y208C. The cells Ž10% of the well content. were removed from the original plates in duplicates, washed 2 = in PBS, and 60 m l of enzyme substrate was added and incubated with the cells for 6 h at 378C. The reaction was stopped by adding 90 m l of 50 mM glycine buffer, pH 10.4 containing 5 mM EDTA. The absorbance was measured at 405 nm in a Microelisa Reader ŽDynatech MR7000..

3. Results 3.1. Characterization of the a-Thy-1-hematoporphyrin conjugate The purified and previously characterized a-Thy-1 rat monoclonal antibody was chemically conjugated through EDCl to the free HP molecules. The method allows the free carboxyl groups of the HP molecules to couple to the free amino groups of the protein molecules. The HP-protein complex was separated from the free HP molecules on a Sephadex G-25 column after freeze-drying, which allows the separation of the non-covalently attached HP molecules

Fig. 1. Flow cytometric analysis of the anti-Thy-1 mAb and its HP conjugate reactivity on T ŽEL-4. and B ŽA20. cell lines detected by indirect labeling Žanti-rat Ig-FITC. method. Both the free mAb Žfine line. and its conjugate Žbold line. showed selective and specific reaction on T cells. The conjugation step did not alter the affinity and specificity of the mAb.

from the protein carrier Žtested by TLC. ŽBelcher et al., 1970.. This absorption of HP molecules to proteins is a characteristic feature of the charged, polar HP molecules ŽKessel and Chou, 1983.. After the separation steps, the average number of covalently bound HP molecules to one a-Thy-1 immunoglobulin molecule was around 30, which reflects a good coupling efficiency. The chemical coupling reaction did not impair the antigen binding ability of the a-Thy-1 antibody, as measured by flow cytometry on B ŽA20. and T ŽEL-4. cells ŽFig. 1.. The labeling of a-Thy-1 mAb with HP did not result in its increased non-specific binding to Thy-1 negative cells. 3.2. In Õitro photosensitizing effect of the antibodyHP conjugate The cytotoxic potential of the conjugate was first tested in vitro on the Thy-1 positive EL-4 cell line and mouse thymocytes and on Thy-1 negative cell lines ŽSp-2r0-Ag14 and a-Insulin.. The cells were treated with the HP-anti-Thy-1 conjugate for 1 h at 48C in the dark. The HP content of the conjugate ranged from 0 to 1.5 m grml HP Ž0–11 m grml mAb. concentrations, and its effects were compared to those of free HP alone at the same concentrations, or to mAb added alone, or culture medium as negative controls. After removing the unbound material with two washing steps, the cells were plated and irradiated Žin triplicates. with a Hg lamp at a wavelength peak of 400 nm at 2–12 Jrcm2 , while the control samples Žlabeled, but not irradiated. were further kept in the dark. Prominent morphological changes could be observed after photo-immunotargeting treatment of the different B and T cell types. The T cells affected displayed irregular shape, shrinkage and fragmented nuclei. In contrast, the B cells appeared to be intact after the selective photosensitization Ždata not shown.. The viability of the cells was tested immediately, 1 h and 24 h after the photo-immunotargeting treatment. The conjugate at 1.26 m grml HP concentration Ž10 m grml mAb. caused 99% EL-4 cell destruction and 96% thymocyte death, measured 1 h later, while the same treatment did not influence the viability of the Thy-1 negative cell types ŽFig. 2.. The free HP at the same

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sured in the same sample. The colorimetric cell proliferation assay showed parallel results with the cell viability: 24 h after the irradiation, the destroyed T cell population did not show significant enzyme activity; while a mild recovery of the surviving 1–2% cells could be measured after 48 h Žweak yellow color. compared to the intense yellow reaction of the unaffected B cell groups ŽTable 1.. The free HP caused only a mild decrease of cell proliferation, while the free mAb alone did not induce significant alterations. The hybridoma cell line and Sp2r0-Ag14 myeloma cells did not show any sign of photosensitization after the same treatment of the conjugate. Fig. 2. Survival curves of different T Žempty symbols. and B Žfilled symbols. cell types after photo-immunotargeting treatment using different doses of the HP-anti-Thy-1 conjugate. 10 m grml antibody Ž1.26 m grml HP. caused 99% cell destruction of EL4 cells and 96 thymocyte death, while the same amount did not alter the viability of the Thy-1 negative ŽSp-2r0, a-Insulin hybridoma. cells as determined 1 h after treatment.

concentration did not influence the cell viability after the same dose of irradiation. Neither the cells treated with the conjugate, but kept in the dark, nor the mAb alone followed by irradiation, had any effect on T cell viability even 24 h after the treatment. The viability of the cells showed differences 1 h and 24 h after the treatment. At lower concentrations Ž5, 2.5 m grml. the conjugate caused only 50–80% cell death when measured 1 h following irradiation, while 24 h later, 90–98% cell destruction could be mea-

3.3. SelectiÕity of the method The selectivity of the method was measured in a mixed cell population, where EL-4 cells and hybridoma cells were mixed at 1:1 ratio, treated with the conjugate at the above-mentioned concentrations andror HP alone, or mAb alone and irradiated with the Hg-lamp at 400 nm wavelength peak. One hour later, the cell viability was measured by trypan blue dye exclusion test, and the cells were examined by flow cytometry with double staining method 24 h later. The B cells were labeled with a-CD24 antibody, followed by anti-rat-FITC, while the T cells were stained with biotin-labeled a-Thy-1 antibody, followed by streptavidin–PE conjugate ŽFig. 3.. The HP-a-Thy-1 mAb treated and irradiated sample Žbold line. was compared to the non-irradiated Žbut treated

Table 1 Landegren colorimetric cell proliferation assay of in vitro cultured EL-4 cells 48 h after photosensitization Treatment Žlight doserconcentration.

HP-a-Thy 1.2 conjugate

HP m grml mAb m grml 0.00 Jrcm2 10.00 Jrcm2

1.26 10.00 2.625a 0.038

a

0.63 5.00 2.497 0.145

0.31 2.50 2.614 0.274

Free HP

Free mAb

Untreated Žmedia only.

1.00 y 2.627 2.291

y 10.00 2.467 2.302

y y 2.639 2.547

Numbers indicate average OD values measured at 405 nm in triplicates. mAb: monoclonal antibody, HP: hematoporphyrin. Concentration-dependent inhibition of cell proliferation was measured with colorimetric assay of the HP-a-Thy-1 mAb treated and irradiated T cell samples. The effects of the same concentration of free HP and the free mAb were compared.

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Fig. 3. Flow cytometric detection of anti-CD24 Žqa-rat FITC. and anti-Thy-1-biotin Žqstr-PE. double-labeled cell mixture 24 h after the photo-immunotargeting treatment. The dot plot of the cell mixture showed two isolated cell populations: R1 contains the hybridoma cells, whereas R2 contains EL-4 cells. In histogram A, the anti-CD24 reactivity of the hybridoma cells ŽG1. in irradiated Žbold. and non-irradiated Žfine. cell mixture was compared and showed similar reaction. Histogram B shows the disappearance of anti-Thy-1 reactivity of T cells ŽG2. after irradiation Žbold. comparing to the strong reaction of non-irradiated Žbut previously HP-anti-Thy-1 treated. Žbold line. samples. Dotted lines represent the secondary antibody controls.

with the conjugate. cell mixture Žsolid line.. The photo-immunotargeting treatment of the cell mixture with our conjugate induced the selective disappearance of the Thy-1 positive cells in the irradiated samples Žbold line on histogram B., while the a-CD24 staining of the B cells did not change after irradiation Žbold line on histogram A.. The free HP at this low concentration, or the irradiation alone did not influence the staining Žviability. or proliferation of cell mixture.

4. Discussion A new method has been developed for the selective destruction of one population Žtarget cell. within an in vitro heterogeneous cell mixture. In our model, Thy-1 antigen bearing cells ŽBarclay et al., 1993. were labeled with a monoclonal antibody–hematoporphyrin conjugate ŽHP-a-Thy-1 mAb. and subsequently irradiated with visible light. The monoclonal

antibody-bound photosensitizer HP molecules ensure selectivity at two levels: the targeting of the HP with an antibody, followed by a local irradiation Žactivation. step. The previously used He–Ne laser irradiation for activating the unbound HP molecules at 632.8 nm wavelength Ž10–20 Jrcm2 . ŽBerki and Nemeth, ´ 1992. was inefficient in this in vitro phototargeting model. The HP molecules bound to proteins changed their absorption properties and decreased, even lost the absorption peak at 630 nm wavelength Ždata not shown.. The highest absorption peak at 400 nm wavelength ŽSoret band. did not change. It is likely that this shift and the relatively small amount of HP molecules bound to each cell might account for the inefficiency. This shift resulted in an effective activation of the mAb-conjugated molecules at a relatively higher photon emission energy at a lower wavelength, as detected by the selective killing Ž99%. of Thy-1 bearing cell types ŽEL-4 and mouse thymocytes.. Irradiation of unlabelled EL-4 cells, or la-

T. Berki, P. Nemethr Journal of Immunological Methods 211 (1998) 139–146 ´

beled Thy-1 negative cell types ŽSp-2r0-Ag14, a-Insulin hybridoma cells. did not induce changes in the viability and proliferation rates of the cell lines. Therefore, the activation of the conjugate at 400 nm seems to be useful for the in vitro application, without noticeable toxic side effects. The conjugate coupling procedure Žcovalent binding by EDCl. must allow high HPrmAb molar substitution ratio Ž30r1 in this case. while preserving the activity of both components. We had to remove the absorbed free HP molecules Žby a freeze-drying step. from the IgG protein molecules that otherwise might have caused unwanted photosensitization by leakage to and uptake by unlabelled components of the in vitro cell mixture. The pure HP-mAb conjugate both retained its binding activity and was selectively phototoxic, killing only those cells that had bound the mAb. In the absence of light, the specifically bound conjugates had no appreciable toxicity. The HP–mAb conjugate binding to the cells was carried out at 48C, and was immediately followed by irradiation, so there should have been no internalization or shedding of the membrane-bound conjugates. Thus, for the HP molecules, it is not necessary to enter the cells to induce photolysis, because the cell membrane is permeable for the free radicals generated during the photosensitization process ŽMao and Poznansky, 1992.. This is consistent with our previous observations with free HP molecules, where changes in the cell membrane fluidity after irradiation were demonstrated in a fluorescence spectroscopic study ŽLakos and Berki, 1995.. The data indicate that the cell membrane seems to be the principal target during the photo-immunotargeting process ŽBerki et al., 1991.. However, the detection of fine molecular changes needs further investigations. The damage process did not immediately alter membrane permeability, since fewer cells showed trypan blue dye uptake at the end of irradiation, then 1 h and 24 h later Ž99% cell death.. This might reflect a similar energy transfer chain reaction resulting in the production of different O 2-derived mediators and free radicals ŽPoto ´ ´ and Berki, 1989; Buettner et al., 1993., previously observed in free HP photosensitization. Since the number of mAbs bound at the cell surface Ž10 6rcell. is known, it is possible to roughly

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quantify the photolytic process ŽOseroff et al., 1987.. Following our spectroscopic measurements, there is an average of 30 HP molecules bound to one mAb molecule, which results in 3 = 10 7 HP molecules per cell Žwhich is 3 = 10y1 4 g of HP per cell. and a light dose of 6 Jrcm2 at 405 nm to kill 98% of the labeled cells. Similar data have been reported by Bellnier and Dougherty Ž1982. using free HPD uptake by in vitro cultured cell lines. They calculated 2.3 = 10y1 3 g of HPD per cell and a light dose of 2.2 Jrcm2 Ž590–650 nm. to kill 90% of the cells, which is comparable to the present work. However, to reach this concentration at the cell surface we need to apply 10–20 times less HP in protein bound form than it is typically used with free HPD for in vitro phototoxicity ŽVever-Bizet and Brault, 1993.. At the same time, it is impossible to selectively sensitize one cell type with free HPD in an in vitro cell mixture ŽYamamoto et al., 1994.. In proteinbound form, the lower dose of photosensitizer HP molecules and the specific binding of the conjugate together make it possible to label and kill selectively one component in an in vitro cell population without the destruction of surrounding, non-labeled cell types. Photo-immunotargeting may have several other advantages ŽPogrebniak et al., 1993. over mAbs used alone ŽKing et al., 1994. or coupled to toxins ŽVitetta and Thorpe, 1987., drugs ŽDevanathan et al., 1990. or radioisotopes ŽSautter-Bihl and Bihl, 1994.. First, since the HP–mAb conjugate does not have intrinsic toxic effect Žonly after a local irradiation step., it will not cause cell destruction after the activation step Žon the cells taking up the conjugate non-specifically.. Second, the conjugate does not need to be internalized because the diffusion distance of the toxic O 2 derivatives ŽMao and Poznansky, 1992. generated during illumination may lead to an effective membrane destruction. However, the mechanism of this pathway, as well as the in vitro pharmacokinetics of the conjugate, need further investigations.

Acknowledgements This work was supported by a grant from the National Science Foundation of Hungary ŽOTKA No. F 6215..

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