單價抗體被認為不管是被動的體內給予，或是藉由送入基因使體內自行產生，在對抗疾病抗原是相當具有潛力的。很多人類癌症具有HER-2/neu高度表現，其中包含乳癌、卵巢癌、膀胱癌、肺癌以及胃癌等。在動物模式實驗中，高度HER-2/neu表現可增加癌症的惡化以及轉移程度。因此，對抗erbB2的單價抗體應該是具有結合，或治療HER-2/neu高度表現癌症的能力。先前研究指出，細胞膜表現抗erbB2單價抗體的細胞，已經可以專一性的和有表現HER-2/neu的細胞作結合。在我們的實驗中，為了使反轉錄病毒可以和HER-2/neu高度表現的癌症作結合，我們將erbB2的單價抗體嵌入病毒外套膜的proline-rich region (PRR)，這個位置曾被報導可以接受一小段胺基酸的嵌入，而不影響病毒顆粒的穩定性。這個被改造的病毒可以藉由erbB2的單價抗體來感染人類膀胱癌細胞，和野生種只能感染小鼠細胞的特性是不同的。另外，被改造的病毒也可以藉由erbB2單價抗體來破壞HER-2/neu的訊息傳遞。在癌症的治療中，儘管原位腫瘤得到控制，具有惡化或是淋巴轉移的腫瘤對全身體來說都是很危險的。而且，腫瘤細胞的轉型通常涉及致癌基因的擴大，以及啟動子的加強。人類腺病毒E1A的基因產物，可以藉由抑制HER-2/neu啟動子活性來達到抑制腫瘤的生長。因此，我們構築一個由erbB2單價抗體改造的反轉錄病毒，以及攜帶E1A治療基因，來實驗是否可以經由破壞HER-2/neu的訊息傳遞，和抑制HER-2/neu啟動子活性，來治療腫瘤。由動物模式的研究結果顯示，這個被改造的病毒確實可以抑制高度HER-2/neu表現的癌症生長，以及增加老鼠的存活率。這個改造病毒的策略，應該可以成為有效的HER-2/neu表現癌症的治療。

　　Antibody single-chain variable fragment (scFv) has considerable therapeutic potential against antigens involved in disease processes either as proteins synthesized ex vivo for passive administration or introduced by gene therapy for in vivo expression. Many human tumors, including breast, ovarian, bladder, lung and gastric tumors, are known to overexpress HER-2/neu (c-erbB2) oncoprotein. In animal models, HER-2/neu overexpression has been shown to enhance malignant and metastatic phenotypes. Anti-erbB2 scFv may have targeting or therapeutic effects on HER-2/neu-overexpressing tumors. Cells expressing a membrane- anchored anti-erbB2 scFv have been constructed, which could recognize and bind specifically to the target cells expressing the erbB2 antigen. In this study, in order to produce retroviral vectors targeting to HER-2/neu-overexpressing tumors, anti-erbB2 scFv was fused to the variable proline-rich region (PRR) of the envelope glycoprotein gp70 of ecotropic Moloney murine leukemia virus. The PRR in the viral envelope has been reported to tolerate a small insertion of peptides, which does not affect the stability of viral particles. The modified retrovirus but not the wild-type virus can infect human bladder tumor cells overexpressing HER-2/neu. The anti-erbB2 scFv on the retrovirus envelope has also been suggested to disrupt ligand-activated signaling and dimerization of HER-2/neu. In cancer treatment, despite local tumor control, patients with locally advanced bladder cancer or lymphogenic metastasized urothelial carcinoma are at risk for systemic progression. Moreover, transformation of tumor cells is involved in oncogene amplification or promoter enhancement. The human adenovirus early-region 1A (E1A) gene products exert strong tumor-suppressive activities in human tumors via transcriptional suppression of HER-2/neu. Thus, we constructed an anti-erbB2 scFv modified retrovirus expressing E1A, and examined whether it exerted antitumor activities via targeting or signal-silence effects of anti-erbB2 scFv and tumor-suppressive effect of E1A. In the animal study, the modified retrovirus could inhibit HER-2/neu-overexpressing bladder tumor growth and enhance mice survival. Such modified retroviruses may therefore be useful in HER-2/neu-overexpressing cancer treatment.

References

Akamatsu,M., Matsumoto,T., Oka,K., Yamasaki,S., Sonoue,H., Kajiyama,Y., Tsurumaru,M., and Sasai,K. (2003). c-erbB-2 oncoprotein expression related to chemoradioresistance in esophageal squamous cell carcinoma. Int. J. Radiat. Oncol. Biol. Phys. 57, 1323-1327.
Battini,J.L., Danos,O., and Heard,J.M. (1995). Receptor-binding domain of murine leukemia virus envelope glycoproteins. J. Virol. 69, 713-719.
Bellmunt,J., de,W.R., Albiol,S., Tabernero,J., Albanell,J., and Baselga,J. (2003). New drugs and new approaches in metastatic bladder cancer. Crit Rev. Oncol. Hematol. 47, 195-206.
Chen,J.S., Lan,K., and Hung,M.C. (2003). Strategies to target HER2/neu overexpression for cancer therapy. Drug Resist. Updat. 6, 129-136.
Chu,T.H. and Dornburg,R. (1997). Toward highly efficient cell-type-specific gene transfer with retroviral vectors displaying single-chain antibodies. J. Virol. 71, 720-725.
Chu,T.H. and Dornburg,R. (1995). Retroviral vector particles displaying the antigen-binding site of an antibody enable cell-type-specific gene transfer. J. Virol. 69, 2659-2663.
Cook,J.L., Miura,T.A., Ikle,D.N., Lewis,A.M., Jr., and Routes,J.M. (2003). E1A oncogene-induced sensitization of human tumor cells to innate immune defenses and chemotherapy-induced apoptosis in vitro and in vivo. Cancer Res. 63, 3435-3443.
Davey,R.A., Zuo,Y., and Cunningham,J.M. (1999). Identification of a receptor-binding pocket on the envelope protein of friend murine leukemia virus. J. Virol. 73, 3758-3763.
De,L.C., Palmer,D.B., Piccoli,R., Ritter,M.A., and D'Alessio,G. (2002). A new human antitumor immunoreagent specific for ErbB2. Clin. Cancer Res. 8, 1710-1719.
Deas,O., Angevin,E., Cherbonnier,C., Senik,A., Charpentier,B., Levillain,J.P., Oosterwijk,E., Hirsch,F., and Durrbach,A. (2002). In vivo-targeted gene delivery using antibody-based nonviral vector. Hum. Gene Ther. 13, 1101-1114.
Dinney,C.P., Fishbeck,R., Singh,R.K., Eve,B., Pathak,S., Brown,N., Xie,B., Fan,D., Bucana,C.D., Fidler,I.J., and . (1995). Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice. J. Urol. 154, 1532-1538.
Flint,J. and Shenk,T. (1989). Adenovirus E1A protein paradigm viral transactivator. Annu. Rev. Genet. 23, 141-161.
Flinterman,M., Gaken,J., Farzaneh,F., and Tavassoli,M. (2003). E1A-mediated suppression of EGFR expression and induction of apoptosis in head and neck squamous carcinoma cell lines. Oncogene 22, 1965-1977.
Graus-Porta,D., Beerli,R.R., Daly,J.M., and Hynes,N.E. (1997). ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J. 16, 1647-1655.
Grooteclaes,M.L. and Frisch,S.M. (2000). Evidence for a function of CtBP in epithelial gene regulation and anoikis. Oncogene 19, 3823-3828.
Heard,J.M. and Danos,O. (1991). An amino-terminal fragment of the Friend murine leukemia virus envelope glycoprotein binds the ecotropic receptor. J. Virol. 65, 4026-4032.
Ishida,T., Tsujisaki,M., Hanzawa,Y., Hirakawa,T., Hinoda,Y., Imai,K., and Yachi,A. (1994). Significance of erbB-2 gene product as a target molecule for cancer therapy. Scand. J. Immunol. 39, 459-466.
Jimenez,R.E., Hussain,M., Bianco,F.J., Jr., Vaishampayan,U., Tabazcka,P., Sakr,W.A., Pontes,J.E., Wood,D.P., Jr., and Grignon,D.J. (2001). Her-2/neu overexpression in muscle-invasive urothelial carcinoma of the bladder: prognostic significance and comparative analysis in primary and metastatic tumors. Clin. Cancer Res. 7, 2440-2447.
Khare,P.D., Shao-Xi,L., Kuroki,M., Hirose,Y., Arakawa,F., Nakamura,K., Tomita,Y., and Kuroki,M. (2001). Specifically targeted killing of carcinoembryonic antigen (CEA)-expressing cells by a retroviral vector displaying single-chain variable fragmented antibody to CEA and carrying the gene for inducible nitric oxide synthase. Cancer Res. 61, 370-375.
Ma,Y., Zhou,X., Zhao,Q., Li,Y., Liu,Y., Wang,Z., and Zhang,Y. (2003). Expression of adenovirus type 5 E1A in the methylotrophic yeast Pachia pastoris and the inhibitory effect on S-180 tumor growth. Biol. Pharm. Bull. 26, 137-140.
Martin,F., Kupsch,J., Takeuchi,Y., Russell,S., Cosset,F.L., and Collins,M. (1998). Retroviral vector targeting to melanoma cells by single-chain antibody incorporation in envelope. Hum. Gene Ther. 9, 737-746.
Mymryk,J.S., Shire,K., and Bayley,S.T. (1994). Induction of apoptosis by adenovirus type 5 E1A in rat cells requires a proliferation block. Oncogene 9, 1187-1193.
Neve,R.M., Sutterluty,H., Pullen,N., Lane,H.A., Daly,J.M., Krek,W., and Hynes,N.E. (2000). Effects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells. Oncogene 19, 1647-1656.
Nguyen,T.H., Loux,N., Dagher,I., Vons,C., Carey,K., Briand,P., Hadchouel,M., Franco,D., Jouanneau,J., Schwall,R., and Weber,A. (2003). Improved gene transfer selectivity to hepatocarcinoma cells by retrovirus vector displaying single-chain variable fragment antibody against c-Met. Cancer Gene Ther. 10, 840-849.
Press,M.F., Cordon-Cardo,C., and Slamon,D.J. (1990). Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene 5, 953-962.
Rao,L., Debbas,M., Sabbatini,P., Hockenbery,D., Korsmeyer,S., and White,E. (1992). The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins. Proc. Natl. Acad. Sci. U. S. A 89, 7742-7746.
Schnierle,B.S. and Groner,B. (1996). Retroviral targeted delivery. Gene Ther. 3, 1069-1073.
Shao,R., Xia,W., and Hung,M.C. (2000). Inhibition of angiogenesis and induction of apoptosis are involved in E1A-mediated bystander effect and tumor suppression. Cancer Res. 60, 3123-3126.
Shinnick,T.M., Lerner,R.A., and Sutcliffe,J.G. (1981). Nucleotide sequence of Moloney murine leukaemia virus. Nature 293, 543-548.
Shou,J., Massarweh,S., Osborne,C.K., Wakeling,A.E., Ali,S., Weiss,H., and Schiff,R. (2004). Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer. J. Natl. Cancer Inst. 96, 926-935.
Somia,N.V., Zoppe,M., and Verma,I.M. (1995). Generation of targeted retroviral vectors by using single-chain variable fragment: an approach to in vivo gene delivery. Proc. Natl. Acad. Sci. U. S. A 92, 7570-7574.
Suzuki,M., Shinkai,M., Honda,H., Kamihira,M., Iijima,S., and Kobayashi,T. (2001). Construction of tumor-specific cells expressing a membrane-anchored single-chain Fv of anti-ErbB-2 antibody. Biochim. Biophys. Acta 1525, 191-196.
Tai,C.K., Logg,C.R., Park,J.M., Anderson,W.F., Press,M.F., and Kasahara,N. (2003). Antibody-mediated targeting of replication-competent retroviral vectors. Hum. Gene Ther. 14, 789-802.
Underwood,M., Bartlett,J., Reeves,J., Gardiner,D.S., Scott,R., and Cooke,T. (1995). C-erbB-2 gene amplification: a molecular marker in recurrent bladder tumors? Cancer Res. 55, 2422-2430.
Vogt,P.K. and Hu,S.S. (1977). The genetic structure of RNA tumor viruses. Annu. Rev. Genet. 11, 203-238.
Weimin,W.B., Cannon,P.M., Gordon,E.M., Hall,F.L., and Anderson,W.F. (1998). Characterization of the proline-rich region of murine leukemia virus envelope protein. J. Virol. 72, 5383-5391.
Yu,D., Suen,T.C., Yan,D.H., Chang,L.S., and Hung,M.C. (1990). Transcriptional repression of the neu protooncogene by the adenovirus 5 E1A gene products. Proc. Natl. Acad. Sci. U. S. A 87, 4499-4503.
Yu,D.H., Scorsone,K., and Hung,M.C. (1991). Adenovirus type 5 E1A gene products act as transformation suppressors of the neu oncogene. Mol. Cell Biol. 11, 1745-1750.

Wu, B. W., Cannon, P. M., Gordon, E. M., Frederick, L. H., and Anderson, W. F. (1998). Characterization of the proline-rich region of murine leukemia virus envelope protein. J. Virol. 72, 5383-5390.