Purpose: Pancreatic carcinoma is highly resistant to therapy. Epidermal growth factor receptor (EGFR) and HER2 have been reported to be both dysregulated in this cancer. To evaluate the in vivo effect of binding both EGFR and HER2 with two therapeutic humanized monoclonal antibodies (mAb), we treated human pancreatic carcinoma xenografts, expressing high EGFR and low HER2 levels. Experimental Design: Nude mice, bearing xenografts of BxPC-3 or MiaPaCa-2 human pancreatic carcinoma cell lines, were injected twice weekly for 4 weeks with different doses of anti-EGFR (matuzumab) and anti-HER2 (trastuzumab) mAbs either alone or in combination.The effect of the two mAbs, on HER receptor phosphorylation, was also studied in vitro by Western blot analysis. Results: The combined mAb treatment significantly inhibited tumor progression of the BxPC-3 xenografts compared with single mAb injection (P = 0.006) or no treatment (P = 0.0004) and specifically induced some complete remissions. The two mAbs had more antitumor effect than 4-fold greater doses of each mAb. The significant synergistic effect of the two mAbs was confirmed on the MiaPaCa-2 xenograft and on another type of carcinoma, SK-OV-3 ovarian carcinoma xenografts. In vitro, the cooperative effect of the two mAbs was associated with a decrease in EGFR and HER2 receptor phosphorylation. Conclusions: Anti-HER2 mAb has a synergistic therapeutic effect when combined with an anti-EGFR mAb on pancreatic carcinomas with low HER2 expression. These observations may open the way to the use of these two mAbs in a large panel of carcinomas expressing different levels of the two HER receptors.The incidence of pancreatic cancer has steadily increased over the past four decades, and its prognosis is still dismal, despite all efforts in early diagnosis and therapy. Even with a complete surgical resection, the 5-year survival rate is <20% (1).Conventional therapies associating surgery and radiotherapy often in combination with chemotherapy show modest efficacy in local control and palliation and no real progress in patient survival (2 -4). Thus, novel approaches to human pancreatic carcinoma therapy are urgently needed.Among the numerous biochemical and genetic abnormalities that were found to be associated with this malignancy (3), we were interested by the fact that two receptors of the HER tyrosine kinase family epidermal growth factor receptor (EGFR; ErbB1 or HER1) and HER2 (ErbB2) are expressed in a significant percentage of cases ranging from 45% to 95% for EGFR (5, 6) and 43% to 69% for HER2 (7,8). The levels of HER2 expression, however, were low in the majority of tumors. Furthermore, the overexpression of EGFR and HER2 receptors is often caused by encoding gene amplification (9).EGFR and HER2 are known to play an essential role in regulating cell proliferation and differentiation. They have a strong tendency to assemble with other HER receptors into homodimers and/or heterodimers on extracellular growth factor binding, which results in various forms of signal transd...

Small recombinant antibody molecules such as bispecific single-chain diabodies (scDb) possessing a molecular mass of ϳ55 kDa are rapidly cleared from circulation. We have recently extended the plasma half-life of scDb applying various strategies including PEGylation, N-glycosylation and fusion to an albumin-binding domain (ABD) from streptococcal protein G. Here, we further analyzed the influence of these modifications on the biodistribution of a scDb directed against carcinoembryonic antigen (CEA) and CD3 capable of retargeting T cells to CEAexpressing tumor cells. We show that a prolonged circulation time results in an increased accumulation in CEA ؉ tumors, which was most pronounced for scDb-ABD and PEGylated scDb. Interestingly, tumor accumulation of the scDb-ABD fusion protein was ϳ2-fold higher compared with PEGylated scDb, although both molecules exhibit similar plasma half-lives and similar affinities for CEA. Comparing half-lives in neonatal Fc receptor (FcRn) wild-type and FcRn heavy chain knock-out mice the contribution of the FcRn to the long plasma half-life of scDb-ABD was confirmed. The half-life of scDb-ABD was ϳ2-fold lower in the knock-out mice, while no differences were observed for PEGylated scDb. Binding of the scDb derivatives to target and effector cells was not or only marginally affected by the modifications, although, compared with scDb, a reduced cytotoxic activity was observed for scDb-ABD, which was further reduced in the presence of albumin. In summary, these findings demonstrate that the extended half-life of a bispecific scDb translates into improved accumulation in antigen-positive tumors but that modifications might also affect scDb-mediated cytotoxicity.Bispecific single-chain diabodies (scDb) 2 are recombinant molecules composed of the variable heavy and light chain domains of two antibodies connected by three linkers in the order V H A-V L B-V H B-V L A (1). These domains assemble into molecules with a compact structure and molecular masses of ϳ55 kDa. Bispecific single-chain diabodies have been developed for various applications including the retargeting of cytotoxic T lymphocytes to tumor cells for cellular cancer therapy (2).Although scDb are capable of efficiently retargeting effector cells to tumor cells the small size leads to their rapid elimination after i.v. injection. The terminal half-life of these molecules in mice is only in the range of 5-6 h, compared with several days for whole IgG molecules (3, 4). The fast clearance of such small molecules from circulation hampers therapeutic applications, e.g. requiring infusions or repeated injections to maintain a therapeutically effective dose over a prolonged period of time (5). For example, a bispecific tandem scFv directed against CD19 and CD3 (blinatumomab) having a similar size as an scDb molecule had to be given as an 8-week infusion (maximum dose 60 g/m 2 per day) in a clinical phase I trial for the treatment of B cell lymphoma patients (6).To extend plasma half-lives of therapeutic proteins and thus to improve ph...

We previously demonstrated the synergistic therapeutic effect of the cetuximab (anti-epidermal growth factor receptor [EGFR] monoclonal antibody, mAb)-trastuzumab (anti-HER2 mAb) combination (2mAbs therapy) in HER2(low) human pancreatic carcinoma xenografts. Here, we compared the 2mAbs therapy, the erlotinib (EGFR tyrosine kinase inhibitor [TKI])-trastuzumab combination and lapatinib alone (dual HER2/EGFR TKI) and explored their possible mechanisms of action. The effects on tumor growth and animal survival of the three therapies were assessed in nude mice xenografted with the human pancreatic carcinoma cell lines Capan-1 and BxPC-3. After therapy, EGFR and HER2 expression and AKT phosphorylation in tumor cells were analyzed by Western blot analysis. EGFR/HER2 heterodimerization was quantified in BxPC-3 cells by time-resolved FRET. In K-ras-mutated Capan-1 xenografts, the 2mAbs therapy gave significantly higher inhibition of tumor growth than the erlotinib/trastuzumab combination, whereas in BxPC-3 (wild-type K-ras) xenografts, the erlotinib/trastuzumab combination showed similar growth inhibition but fewer tumor-free mice. Lapatinib showed no antitumor effect in both types of xenografts. The efficacy of the 2mAbs therapy was partly Fc-independent because F(ab')(2) fragments of the two mAbs significantly inhibited BxPC-3 growth, although with a time-limited therapeutic effect. The 2mAbs therapy was associated with a reduction of EGFR and HER2 expression and AKT phosphorylation. BxPC-3 cells preincubated with the two mAbs showed 50% less EGFR/HER2 heterodimers than controls. In pancreatic carcinoma xenografts, the 2mAbs therapy is more effective than treatments involving dual EGFR/HER2 TKIs. The mechanism of action may involve decreased AKT phosphorylation and/or disruption of EGFR/HER2 heterodimerization.