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Drug Candidate: BreMel/rGel (scFvMel/rGel) (single-chain immunotoxin) Indications: Breast Cancer and Melanoma

Stage: Pre-clinical Overview: BreMel/rGel is currently being preclinically developed for the treatment and prophylaxis of metastatic melanoma and a molecularly-defined subset of breast cancers. BreMel/rGel (aka scFvMel/rGel) is an unglycosylated, low molecular weight (46 kDa) recombinant fusion protein. BreMel/rGel consists of a single chain antibody fragment (scFv) genetically fused to the toxin payload rGel (recombinant gelonin). It can be readily manufactured inexpensively and efficiently in E. coli fermenters. The construct uses a small flexible linker between the variable domains of the heavy and light chains of the antibody ZME-018 attached via a tether (G4S) to the recombinant gelonin. This molecule has the binding properties of the original anti-melanoma antibody and contains biologically active toxin. The original antibody binds to a surface glycoprotein (gp240) found on over 80% of human melanoma cell lines and biopsy specimens. While most melanomas are currently treated surgically, metastatic disease with a highly negative prognosis remains a significant risk for such patients. New agents for both the prophylaxis and treatment of metastatic melanoma are currently needed. Recent studies also have demonstrated that this antigen is expressed on 67% (44/66) of lobular breast carcinomas studied. Lobular breast carcinomas account for about 15% of all breast cancers and appear to be an immunologically distinct subclass from HER2+ breast cancers. Pre-clinical studies on A-375 Melanoma cell lines and A-375 tumor bearing nude mice strongly indicate that BreMel/rGel has potent cytotoxic activity both in vitro and in vivo and is an excellent candidate for clinical development. BreMel/rGel also has a sister molecule, BreMel/TNF-alpha (aka scFvMel/TNF), which uses human TNF-alpha instead of recombinant gelonin as its payload. Although not as far developed clinically, BreMel/TNF-alpha is also showing very good activity in animal models. Targa has completed most pre-clinical development on BreMel/rGel and expects to file an IND for this compound within 12 months. References

Targa Therapeutics Corp.

Gary Aronson, CEO [email protected] 11585 Sorrento Valley Road Suite 108 San Diego CA 92121 USA Direct Tel: 1-858-488-1288 Fax: 1-858-488-6288 www.TargaTherapeutics.com

Design, expression, purification, and characterization, in vitro and in vivo, of an antimelanoma single-chain Fv antibody fused to the toxin gelonin. Cancer Res. 2003 Jul 15;63(14):3995-4002. Rosenblum MG, Cheung LH, Liu Y, Marks JW 3rd. Comparative cytotoxicity and pharmacokinetics of antimelanoma immunotoxins containing either natural or recombinant gelonin. Cancer Chemother Pharmacol. 1999;44(4):343-8. Rosenblum MG, Marks JW, Cheung LH. Cellular resistance to the antimelanoma immunotoxin ZME-gelonin and strategies to target resistant cells. Cancer Immunol Immunother. 1996 Feb;42(2):115-21. Rosenblum MG, Cheung L, Kim SK, Mujoo K, Donato NJ, Murray JL. Pharmacokinetics, tissue distribution, and in vivo antitumor effects of the antimelanoma immunotoxin ZME-gelonin. Cancer Immunol Immunother. 1995 May;40(5):339-45. Mujoo K, Cheung L, Murray JL, Rosenblum MG.

A specific and potent immunotoxin composed of antibody ZME-018 and the plant toxin gelonin. Mol Biother. 1991 Mar;3(1):6-13. Rosenblum MG, Murray JL, Cheung L, Rifkin R, Salmon S, Bartholomew R.

2005 AACR Abstracts (687) The anti-gp240 fusion toxin scFvMEL/TNF shows potent antitumor activity and synergy with chemotherapeutic agents Yuying Liu, Weihe Zhang, Lawrence H. Cheung, Qingping Wu, Chun Li, Michael G. Rosenblum. UT M.D. Anderson Cancer Center, Houston, TX The recombinant fusion construct scFvMEL/TNF is composed of the human cytokine tumor necrosis factor-alpha (TNF) and a single-chain Fv recognizing gp240 present on 80 % of melanoma cell lines and fresh tumor samples. Previous studies have characterized

Targa Therapeutics Corp.

Gary Aronson, CEO [email protected] 11585 Sorrento Valley Road Suite 108 San Diego CA 92121 USA Direct Tel: 1-858-488-1288 Fax: 1-858-488-6288 www.TargaTherapeutics.com

the fusion construct demonstrating specific binding to gp240 antigen-positive melanoma cells and retained biological activity of TNF. The fusion protein scFvMEL/TNF was more cytotoxic to antigen-positive A375 melanoma cells than TNF alone and, additionally, was active against AAB-527 melanoma cells completely resistant to TNF itself. Co-administration of scFvMEL/TNF and chemotherapeutic agents (vincristine, etoposide, cisplatin, 5-FU and adriamycin) to A375 cells for 72 hours demonstrated synergistic antitumor activity with adriamycin, or 5-FU and additive effects in combination with vincristine, etoposide or cisplatin. Radiolabeled scFvMEL/TNF was administered to nude mice bearing human melanoma (A375) xenografts, and mice were sacrificed at 24, 48 and 72 h after administration. We found that kidney and spleen contained the highest tissue: blood ratio of all normal organs. At 24 hr after administration, kidney, spleen and liver also contained the highest concentration of drug/gram tissue weight, although these concentrations declined over time. In contrast to normal organs, both the concentration of the label in tumor as well as the tissue: blood ratio of tumor tissue increased over time. Tumor was the highest site of accumulation by 72 hr after administration (% ID/g, 0.158; tumor-to-blood ratio, 7.6 ± 2.2), followed by kidney (% ID/g, 0.089; tissue-to-blood ratio, 5.1 ± 0.2), spleen (% ID/g, 0.049; tissue-toblood ratio, 2.6 ± 0.8) and liver (% ID/g, 0.042; tissue-to-blood ratio, 1.6 ± 0.8). Studies in mice showed an MTD of 5.0 mg/kg with iv administration on a daily X 5 schedule. We stably transfected A375 cells with enhanced green fluorescent protein (GFP) and further examined the antitumor effects of scFvMEL/TNF using A375GFP xenograft tumors growing subcutaneously. We monitored the efficacy of therapy using a Xenogen IVIS 200 Imaging System which allows intravital imaging of transfected cells. Our preliminary data showed that treatment of scFvMEL/TNF at half-MTD doses demonstrated potent antitumor activity and complete tumor regression. Meanwhile, studies of antitumor efficacy evaluated by imaging and caliper measurement will be reported. These studies suggest that scFvMEL/TNF fusion construct may therefore have a significant potential for treatment of melanoma and some breast tumors. Research conducted, in part, by the Clayton Foundation for Research. (6146) The antimelanoma fusion toxin GrB/scFvMEL operates exclusively through direct activation of cellular apoptosis: in vitro and in vivo studies. Yuying Liu, Weihe Zhang, Ting Niu, Lawrence H. Cheung, Michael G. Rosenblum. UT M.D. Anderson Cancer Center, Houston, TX The fusion construct GrB/scFvMEL is composed of the human pro-apoptotic serine protease Granzyme B (GrB) and the single-chain antibody scFvMEL recognizing the gp240 antigen present on melanoma and certain breast tumors. We examined the expression of gp240 antigen on different melanoma cells by using ELISA and flow

Targa Therapeutics Corp.

Gary Aronson, CEO [email protected] 11585 Sorrento Valley Road Suite 108 San Diego CA 92121 USA Direct Tel: 1-858-488-1288 Fax: 1-858-488-6288 www.TargaTherapeutics.com

cytometry. The antigen was present on A375, TXM18L, TXM13 and MEL526 cells; however, TXM1 displayed low-level expression. GrB/scFvMEL bound specifically to antigen expressing cells as detected by an anti-scFvMEL antibody. The fusion construct demonstrated impressive cytotoxic effect against A375, MEL526, TXM18 and TXM13 (I.C.50 20 - 100 nM) and minimal cytotoxicity to TXM1 cells at doses of up to 1 mM. By comparison, the cytotoxic effects were approximately the same as that of another fusion toxin MEL scFv/rGel on these melanoma cells. Because apoptosis controls tumor growth and metastatic spread as well as response to chemotherapeutic agents, we examined the combination of GrB/scFvMEL with chemotherapeutic agents. GrB/scFvMEL demonstrated synergistic antitumor activity with doxorubicin (DOX), vincristine or cisplatin and additive effects in combination with etoposide or cytorabine. The effects of chemotherapeutic agents could be sensitized by pretreatment with GrB/scFvMEL for 6 h on antigen positive target cells. We established A375 DOX resistant subline (A375DR) that displays > 200 fold resistance to DOX compared to parental A375, however, the cytotoxicity of the fusion construct against A375DR (IC50= 60 nM) was approximately the same as that against parental A375 (IC 50= 20 nM). Treatment of A375DR cells with GrB/scFvMEL abrogated Matrigel migration of cells suggesting that this agent may have an impact on metastatic spread. Mice bearing established A375 tumors were treated (i. v. tail vein) with either GrB/scFvMEL (37.5 mg/kg) or saline. The saline treatment group tumors increased 24 fold (from 50 mm3 to 1200 mm3) over 28 days. In contrast, GrB/scFvMEL treated tumors increased 4 fold (from 50 mm3 to 200 mm3). Most tumor cells displayed apoptotic nuclei as assessed by TUNEL assay. Localization of the construct was observed in tumor tissue as assessed by immunohistochemical staining detected using either anti-GrB or anti-scFvMEL antibody. GrB/scFvMEL fusion construct demonstrates impressive antitumor activity, enhances cellular sensitivity to chemotherapy and directly affects metastatic potential of melanoma cells. Targeted delivery of human pro-apoptotic GrB to tumor cells may therefore have a significant potential for cancer treatment. Research conducted, in part, by The Clayton Foundation for Research. 2003 AACR Abstracts In-vitro studies comparing the recombinant, single-chain immunotoxins scFv23/rGel and ML3.9/rGel which recognize the HER2/neu proto-oncogene.

J. W. Marks, L. Cheung, J. D. Marks, Michael G. Rosenblum. UT M. D. Anderson Cancer Center, Houston, TX; University of California San Francisco, San Francisco, CA.

Targa Therapeutics Corp.

Gary Aronson, CEO [email protected] 11585 Sorrento Valley Road Suite 108 San Diego CA 92121 USA Direct Tel: 1-858-488-1288 Fax: 1-858-488-6288 www.TargaTherapeutics.com

The single-chain antibody scFv23, derived from a murine Mab, recognizes the HER2/ neu cell surface domain, as does the human, phage-display-derived single-chain antibody ML3.9 (originally scFvC6.5, J. D.Marks). Genes for both were fused to the gene encoding the recombinant toxin rGelonin (rGel) using PCR. Each fusion construct was then ligated into a bacterial expression plasmid (pET32) for protein synthesis. Induction of bacterial expression resulted in the generation of new and soluble proteins at the expected molecular weights of 58 kDa (scFv23/rGel), and 56 kDa (scFvML3.9/rGel). As assessed by SDS-PAGE, both proteins were purified to homogeneity using IMAC and both were Western-positive for gelonin. A cell-free protein synthesis assay showed that both proteins have inhibitory activity similar to that of the native toxin (I.C.50 of 53 pM, 92 pM and 60 pM for the scFv23/rGel, ML3.9/rGel and rGel respectively), suggesting that no loss of toxin activity occurred in the fusion molecules. ELISA assays showed that both fusion constructs specifically bound to HER2/ neu-positive cells compared to recombinant gelonin. Cytotoxicity studies against antigen-positive SK-BR-3 cells in log phase culture demonstrate that both fusion constructs have I.C.50 values of 21.6 nM and 15.6 nM, which were at least 100-fold lower than free rGel (I.C.50 of 2213 nM), compared with approximately identical values using antigen-negative ME-180 cells (I.C. 50 of 240 nM,1160 nM and 627 nM, respectively). Both constructs retain the specificity of the original antibody, as well as the biological activity of the original rGelonin toxin. Our data suggests that that immunotoxins derived from phage-generated antibodies appear to be equivalent to immunotoxins containing recombinant constructs derived from existing antibodies. Further in vitro and in vivo studies are planned.

9/15/2005 4:11 PM GDA

Targa Therapeutics Corp.

Gary Aronson, CEO [email protected] 11585 Sorrento Valley Road Suite 108 San Diego CA 92121 USA Direct Tel: 1-858-488-1288 Fax: 1-858-488-6288 www.TargaTherapeutics.com

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