The Study Of Killing Effect Of Photofrin-Herceptin Immunoconjugate On Tumor Cell Lines In Vitro

[Objectives] To study the specific killing effect of the Photofrin-Herceptin immunoconjugate on the tumor cells expressing HER-2(+), which will then establish a foundation for further photoimmunotherapy.[Methods]1. SK-BR-3 MCF-7 A549 cell lines were cultured, then the HER-2 expression rates on the surface of these cell lines were identified via FCM.2. Paper chromatography was used to identify the possibility of using ultrafilitation to purify Herceptin.3. After pretreated with ultrafilitation, Herceptin (trastuzumab) was covalently coupled to Photofrin (porfimer sodium) via [l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride] (EDCI), then the immunoconjugate was identified with thin layer chromatography and attempted to be purified in several different ways.4. The immunoactivity of the immunoconjugate was tested by ELISA, then by MTT method to test its killing effect on SK-BR-3, MCF-7 and A549 cell lines respectively which have been reported to express HER-2 on different levels.[Results ]1. All these three cell lines, SK-BR-3/MCF-7/A549, growed well during culture process and no obvious differeces were observed compared with the culture pictures published by ATCC. The HER-2 expression rates on the surface of these cell lineswere 99.04%, 7.55%, 0.44% respectively tested by FCM.2. At Rf=0 before ultrafilition, the value of ~131I-Herceptin, which was very close to the total hold-back rate of 78.2%, was 77.1% demonstrating that almost all ~131I-labelled Herceptin had been retained. The total radioactivity was 2.2% when Rf=0.1 or 0.2 because some small molecules might have been labelled with ~131I. The results of chromatography following ultrafilition showed that the value of Rf=0 was 96.8% and proved to be rather satisfied. Having compared two different layer chromatophy results, we found that most contents in the filtrate were free I and ~131I-labelled some molecules and most macro-molecules had been separated successfully.3. The thin layer chromatography results showed that Herceptin stayed in original position appearing colorless in incondescent light and blue in ultralet light and Rf value was close to zero. In the incondescent light, Photofrin displayed a long red band from original place to 10cm ahead with much redder in distance. Most of the immunoconjugate also displayed in origin with blue color in ultralet light and red color in incondescent light.4. The chromatography with Sephadex G-25 could not be applied to separate free Photofrin from the immunoconjugate because Sephadex G-25 could react with Photofrin during loading and with ethanol during CIP. The purification process of Photofrin with ultralifition revealed that Photofrin could not penetrate through the ultrafiltration membrane of 30,000 MW. Just like the behavior of protein, Photofrin could precipitate in saturated ammonium sulfate solution and redissolved in ethanol solution. The result from affinity chromatography showed that proteinG could react with Photofrin readily in eluent and give off gas.5. The result of enzyme linked immunosorbent assay (ELISA) indicated that the immunoactivity of the immunoconjugate was about 50% weaker than that ofHerceptin.6. The killing effect of Photofrin-Herceptin immunoconjugate on SK-BR-3 cell line in our experimental system proved to be stronger than that of Photofrin, Herceptin or mixture of Photofrin and Herceptin evidently at the same dose (p<0.05) . Herceptin showed scarcely any killing effect but arresting the cells growth. The killing effect of Photofrin (P) was equal to that of Photofrin+Herceptin (P+H), which meaned that Photofrin was the only active component and Herceptin did not compromise the effect of it. The IC50 s of Photofrin-Herceptin immunoconjugate and mixture of Photofrin and Herceptin were 1.5//g /ml and 6.5/zg /ml respectively and their ratio was 0.23.The killing effect of Photofrin-Herceptin immunoconjugate on MCF-7 cell line was slightly stronger than that of Photofrin, Herceptin or mixture of Photofrin and Herceptin (p<0.05) . Herceptin showed no killing effect on this cell line and no influences on its growth.The IC50 s of Photofrin-Herceptin immunoconjugate and mixture of Photofrin and Herceptin were 6.00^g /ml and 6.6/^g /ml respectively and their ratio was 0.91.The killing effect of Photofrin-Herceptin immunoconjugate on A549 cell line was similar to that of Photofrin or mixture of Photofrin and Herceptin (p>0.05) , but stronger than that of Herceptin (p<0.05). Herceptin has not any killing effect on this cell line and no influences on its growth.The IC50 s of Photofrin-Herceptin immunoconjugate and mixture of Photofrin and Herceptin were 5.00^g /ml and 5.00/ig /ml approximately and their ratio was near to 1.00. [Conclusions]1. The HER-2 expression rates on the surface of SK-BR-3 , MCF-7> A549 cell lines fufilled the request of the experiment.2. It was effective and receivable of ultrafiltration to purify Herceptin.3. The immunoactivity of the conjugate was weaker than that of Herceptin. This conjugate had stronger killing effect on cell lines of HER-2(+) than Photofrin, Herceptin or mixture of Photofrin and Herceptin, and the effect on cell lines of HER-2(-) was similar to that three.4. It was difficult to purify Photofrin-Herceptin immmunoconjugate because of complicated physical and chemical characters of Photofrin.