Mathias Schmidt

Recombinant antibody toxins specific for ErbB2 and EGF receptor inhibit the in vitro growth of human head and neck cancer cells and cause rapid tumor regression in vivo

Overexpression of the ErbB2 and epidermal growth factor receptor (EGFR) tyrosine kinases is frequently observed in squamous cell carcinomas of the head and neck, and has been correlated with shorter overall survival. By immunoblot analysis, we have found EGFR and ErbB2 expression in 6 out of 6 established head and neck cancer cell lines. Elevated EGFR protein levels were noted in 3 and elevated ErbB2 levels in 5 of them. Significant expression of EGFR and ErbB2 was also detected in 17 of 47 and 26 of 45 primary tumor samples. Due to their enhanced expression on the tumor cell surface, these receptors can be regarded as suitable targets for directed cancer therapy. We have analyzed the antitumoral activity of recombinant single‐chain antibody toxins specific for ErbB2 and EGFR against head and neck cancer cells in vitro and in vivo. The recombinant toxins consist of the variable domains of the heavy and light chains of monoclonal antibodies (MAbs) genetically fused to a truncated Pseudomonas exotoxin A (ETA). At low concentrations, the ErbB2‐specific single‐chain antibody (scFv) toxin scFv(FRP5)‐ETA and the EGFR‐specific toxins scFv(225)‐ETA and scFv(14E1)‐ETA inhibited the in vitro growth of established head and neck cancer cell lines and primary tumor cells. In a nude mouse tumor model, intratumoral injection of the antibody toxins resulted in the rapid regression of subcutaneously growing CAL 27 tumor xenografts, with scFv(FRP5)‐ETA and scFv(14E1)‐ETA treatment being most effective and leading to the cure of up to 50% of the animals. Our results suggest that EGFR and ErbB2‐specific antibody toxins may become valuable therapeutic reagents for the treatment of squamous cell carcinomas of the head and neck. Int. J. Cancer 86:269–275, 2000.

Regression of cutaneous tumor lesions in patients intratumorally injected with a recombinant single-chain antibody-toxin targeted to ErbB2/HER2

ScFv(FRP5)-ETA is a recombinant single-chain antibody-toxin with binding specificity for ErbB2/HER2. Previously potent antitumoral activity of the molecule against ErbB2 overexpressing tumor cells was demonstrated in vitro and in animal models. Here we report on the first application of scFv(FRP5)-ETA in human cancer patients summarizing case reports collected in four different clinical centers. Eleven patients suffering from metastatic breast and colorectal cancers and from malignant melanoma were treated on a compassionate-use basis by intratumoral injection of scFv(FRP5)-ETA into cutaneous lesions once daily for 7–10 days. Total daily doses ranged from 60 to 900 µg, and total doses per treatment cycle ranged from 0.6 to 6.0 mg. Treatment caused injected tumors to shrink in six of the 10 cases evaluated (60%). Complete regression of injected tumor nodules was accomplished in four patients (40%) and partial reduction in tumor size in another two patients (20%). Adverse reactions were restricted to local symptoms such as pain and inflammation at injection sites which were fully reversible. Only in one patient treated at the highest daily doses systemic liver toxicity of grade 2 was observed and treatment was discontinued on day 7. No hematologic, renal, and/or cardiovascular toxicities were noted. Our results demonstrate that local therapy with scFv(FRP5)-ETA can be effective against ErbB2 expressing tumors justifying further clinical development of this reagent.

Suppression of metastasis formation by a recombinant single chain antibody-toxin targeted to full-length and oncogenic variant EGF receptors.

Cytotoxic strategies which are directed to tumor-associated antigens might be most beneficial for cancer patients with minimal tumor load such as in an adjuvant setting after initial therapy. We have recently described a highly potent single chain antibody-toxin, scFv(14E1)-ETA, which consists of the variable domains of the antibody 14E1 genetically fused to a truncated form of Pseudomonas exotoxin A. ScFv(14E1)-ETA specifically recognizes the human epidermal growth factor receptor (EGFR) and the oncogenically activated receptor variant EGFRvIII, which have been implicated in the development of various human malignancies. Here we have investigated the antimetastatic activity of bacterially expressed scFv(14E1)-ETA and its disulfide-stabilized derivative ds-scFv(14E1)-ETA in a novel model for disseminated disease which is based on murine renal carcinoma cells subsequently transfected with the E. coli β-galactosidase gene, and human full-length or variant EGFR cDNAs. Intravenous injection of these Renca-lacZ/EGFR and Renca-lacZ/EGFRvIII cells in syngenic Balb/c mice led to the formation of pulmonary metastases which were readily detectable upon excision of the lungs and X-gal staining. Systemic treatment of mice with scFv(14E1)-ETA resulted in the complete suppression of Renca-lacZ/EGFRvIII metastasis formation and drastically reduced the number of pulmonary Renca-lacZ/EGFR tumor nodules. The ds-scFv(14E1)-ETA derivative where the antibody variable regions are connected by an artificial disulfide bond displayed improved thermal stability at physiological temperature but due to reduced cytotoxic activity was less potent than the original scFv(14E1)-ETA in metastasis suppression.

A bivalent single‐chain antibody‐toxin specific for ErbB‐2 and the EGF receptor

ErbB‐2 and EGF receptors are often co‐expressed in human tumors and have been shown to synergize in the transformation of cells in experimental model systems. Transactivation of ErbB‐2 can occur via ligand‐induced heterodimerization with EGF receptor or other members of the ErbB family of receptor tyrosine kinases. We have previously described the potent anti‐tumoral activity of the monospecific single‐chain antibody‐toxins scFv(FRP5)‐ETA and scFv(225)‐ETA binding to, respectively, ErbB‐2 and the EGF receptor. Here we report the construction and functional characterization of a novel bivalent, bispecific single‐chain antibody‐toxin, scFv2(FRP5/225)‐ETA. The fusion protein consists of 2 scFv domains specific for ErbB‐2 and the EGF receptor linked to a modified Pseudomonas exotoxin A. ScFv2(FRP5/225)‐ETA displayed in vitro cell killing activity on tumor cells overexpressing either ErbB‐2 or the EGF receptor similar to that of the monospecific toxins. It was more potent in vitro and in vivo in inhibiting the growth of tumor cells expressing both receptors. Treatment of A431 cells with scFv2(FRP5/225)‐ETA led to an increase in EGF receptor and ErbB‐2 phosphotyrosine content, most likely via the induction of receptor heterodimers. This may explain the enhanced toxicity of the bispecific antibody‐toxin.

BIOTECHNOLOGICAL AND GENE THERAPEUTIC STRATEGIES IN CANCER TREATMENT

New anti-cancer agents are being developed which incorporate cancer-cell-specific recognition functions and are thus able to distinguish between normal and tumor cells. Recognition is dependent on the enhanced expression of antigenic determinants on the surface of tumor cells. The ErbB-2 receptor (ErbB-2R) is overproduced in a high percentage of adenocarcinomas arising in the breast, ovary, lung and stomach, when compared to normal cells. The tumor-enriched expression and extracellular accessibility make this receptor a suitable target for directed tumor therapy. A gene expressing the single-chain antibody molecule (scFv), specific for the extracellular domain of the ErbB-2R, was constructed by joining cDNAs encoding the light- and heavy-chain variable domains of the monoclonal antibody (mAb) FRP5. This scFv-encoding gene has been used as a targeting domain for two effectors: (i) A recombinant immunotoxin-encoding gene was constructed by adding sequences encoding a modified Pseudomonas aeroginosa exotoxin A (ETA) to the scFv-encoding DNA. (ii) Cytotoxic T-lymphocytes (CTL) with specificity for ErbB-2R-producing tumor cells were generated by retroviral transfer of a chimeric gene which encodes the scFv(FRP5), a hinge region and the zeta-chain of the T-cell receptor (TCR) complex. The bacterially produced recombinant immunotoxin scFv(FRP5)-ETA binds specifically to the ErbB-2R and displays both in vitro and in vivo cytotoxic effects selective for tumor cells producing high levels of the ErbB-2R. Target cells expressing the ErbB-2R gene were lysed in vitro with high specificity by the scFv::hinge::zeta-expressing T-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

TARGETED THERAPY OF SCHWANNOMA CELLS IN IMMUNOCOMPETENT RATS WITH AN erbB2-SPECIFIC ANTIBODY-TOXIN

Over‐expression of the erbB2‐receptor tyrosine kinase is frequently observed in many human tumors of epithelial origin. Due to its causal involvement in malignant transformation and its presence on the tumor cell surface erbB2 is an attractive target for directed tumor therapy. We earlier described the potent anti‐tumoral activity of the recombinant single‐chain antibody toxin scFv(FRP5)‐ETA in vitro and in nude mouse tumor models in vivo. This molecule consists of the variable domains of the heavy and light chains of an erbB2‐specific antibody genetically fused to a truncated Pseudomonas exotoxin A. Here we have investigated the in vivo effects of this immunotoxin on erbB2 expressing NV2Cd schwannoma cells growing as s.c. tumors in syngeneic BDIX rats. Established tumors were treated either locally by intratumoral injection of scFv(FRP5)‐ETA or systemically by injection into the tail vein. Both routes of application resulted in pronounced inhibition of tumor growth with local treatment being more effective. Treatment with 25 μg/day of scFv(FRP5)‐ETA for 10 days suppressed tumor growth almost completely. Antibodies directed mainly against the toxin domain of the fusion protein developed in all animals treated. Int. J. Cancer 73:117–124, 1997.

Activation of EGF receptor family members suppresses the cytotoxic effects of tumor necrosis factor-α

Abstract Tumor necrosis factor (TNF)-α has a broad range of biological activities, which depend heavily on cell type and physiological condition. In a panel of human tumor cell lines we analyzed expression of the receptor tyrosine kinases EGFR, ErbB2 and ErbB3, and the response to TNF-α. Among the cell lines tested those resistant to TNF-α were found to express high levels of either EGFR, or ErbB2 and ErbB3. In TNF-sensitive breast and cervical carcinoma cells activation of EGFR or ErbB2 by the exogenous growth factors EGF and heregulin β1 resulted in a significant increase in the number of cells surviving TNF-α treatment. In contrast, inhibition of EGFR activation in TNF-resistant breast carcinoma cells by the novel antagonistic anti-EGFR antibody 14E1 sensitized the cells to the cytotoxic effects of TNF-α. A bacterially expressed fusion protein consisting of a 14E1 single-chain (sc) Fv antibody fragment linked to human TNF-α retained TNF-α activity. This scFv(14E1)-TNF-α molecule localized specifically to EGFR on the surface of tumor cells and activated the NF-κB pathway in co-cultured T cells, as demonstrated by electrophoretic mobility shift assays.

Molecular alterations after Polo-like kinase 1 mRNA suppression versus pharmacologic inhibition in cancer cells

Multiple roles within mitosis have been assigned to Polo-like kinase 1 (Plk1), making it an attractive candidate for mitotic targeting of cancer cells. We have employed chimeric antisense oligonucleotides to investigate the molecular alterations after targeted interference with Plk1 in RKO human colon adenocarcinoma and PC3 prostate cancer cells. Suppression of Plk1 mRNA resulted in a dramatic increase of the mitotic index followed by the onset of apoptosis. Mitotically arrested cells displayed randomly separated condensed chromosomes and the occurrence of multiple spindle poles with well-formed asters. Induction of apoptosis was strictly dependent on cell cycle progression: Genetically engineered RKO cells with inducible expression of the cyclin-dependent kinase inhibitor p27Kip1 were completely refractory to Plk1 depletion-induced apoptosis when they were arrested in the G1 phase of the cell cycle. Various mitotic markers, including MPM-2, cdc25c, cyclin B1, or phosphorylated histone H3, were investigated to explore the molecular consequences of Plk1 depletion. Whereas most marker proteins showed similar alterations compared with treatment with paclitaxel, cdc25c was fully phosphorylated solely in paclitaxel-treated cells but only partially phosphorylated in Plk1-depleted cells, although both treatments caused a profound mitotic arrest. This differential phosphorylation of cdc25c was used to test whether a pharmacologic inhibitor of Plk1 would exert the same cellular effects as interference with Plk1 on a mRNA level. It was found that the differential electrophoretic mobility of cdc25c can serve as a reliable molecular marker to track inhibition of Plk1 by small-molecule inhibitors within a cell. [Mol Cancer Ther 2006;5(4):809–17]

Intra-tumoral application of a heregulin-exotoxin-a fusion protein causes rapid tumor regression without adverse systemic or local effects.

Tumor toxins are recombinant, bifunctional proteins which comprise a tumor‐cell‐specific recognition domain and an enzymatic toxin domain. We have evaluated the in vivo effects of a tumor toxin that specifically recognizes the erbB‐3 and erbB‐4 receptors (HRGβI‐ETA). High doses of HRGβI‐ETA administered systemically (intracardially or intraperitoneally) caused acute liver necrosis and were lethal. The same dose of tumor toxins applied subcutaneously had no detectable histopathological effects. The anti‐tumor activity of HRGβI‐ETA was tested in nude mice with xenografts of a human breast tumor, MAXFII62. The MAXFII62 tumor grew rapidly upon s.c. implantation. Intra‐tumoral application of HRGβI‐ETA (7 times 5 μg over a period of 21 days) induced complete regression of tumors. At the time the treatment was terminated, no tumor cells were detectable microscopically. Evaluation of the liver of treated animals revealed no significant toxicity in the effective dose range. These experiments indicate that tumor toxins can become valuable for local tumor treatment and for reduction of tumor burden. Int. J. Cancer 70:682–687, 1997.

Cell type–dependent effects of Polo-like kinase 1 inhibition compared with targeted polo box interference in cancer cell lines

Multiple critical roles within mitosis have been assigned to Polo-like kinase 1 (Plk1), making it an attractive candidate for mitotic targeting of cancer cells. Plk1 contains two domains amenable for targeted interference: a kinase domain responsible for the enzymatic function and a polo box domain necessary for substrate recognition and subcellular localization. Here, we compare two approaches for targeted interference with Plk1 function, either by a Plk1 small-molecule enzyme inhibitor or by inducible overexpression of the polo box in human cancer cell lines. Inducible expression of the Plk1 polo box resulted in growth inhibition of RKOp27 human colon adenocarcinoma cells without obvious signs of mitotic abnormalities. A Plk1 kinase inhibitor in the same cell line arrested cells in mitosis with subsequent onset of apoptosis. Similarly, PC-3 human prostate cancer cells were growth inhibited on expression of the polo box. Prolonged expression of the polo box in these cells resulted in the occurrence of binucleated or multinucleated cells. In contrast, U2OS human osteosarcoma cells responded to overexpression of the polo box with a massive mitotic accumulation coinciding with the onset of apoptosis. Comparison of spindle formation revealed very similar mitotic abnormalities in polo box–overexpressing U2OS cells compared with U2OS cells treated with the Plk1 kinase inhibitor. We conclude that interference with polo box function and inhibition of Plk1 kinase activity can exert very similar phenotypic effects in certain cell lines but highly contrasting effects in others. This may point to subtle differences in the molecular machinery of mitosis regulation in cancer cells. [Mol Cancer Ther 2007;6(12):3189–97]