Klara Totpal

Mapping of the C1q Binding Site on Rituxan, a Chimeric Antibody with a Human IgG1 Fc

Rituxan (Rituximab) is a chimeric mAb with human IgG1 constant domains used in the therapy of non-Hodgkin’s B cell lymphomas. This Ab targets B cells by binding to the cell-surface receptor, CD20. In our investigation of the mechanism of B cell depletion mediated by Rituximab, we first constructed mutants of Rituximab defective in complement activation but with all other effector functions intact. Our results demonstrate that the previously described C1q binding motif in murine IgG2b constituting residues E318, K320, and K322 is not applicable to a human IgG1 when challenged with either human, rabbit, or guinea pig complement. Alanine substitution at positions E318 and K320 in Rituximab had little or no effect on C1q binding and complement activation, whereas alanine substitution at positions D270, K322, P329, and P331 significantly reduced the ability of Rituximab to bind C1q and activate complement. We have also observed that concentrations of complement approaching physiological levels are able to rescue >60% of the activity of these mutant Abs with low affinity for C1q. These data localize the C1q binding epicenter on human IgG1 and suggest that there are species-specific differences in the C1q binding site of Igs.

Engineered Antibodies with Increased Activity to Recruit Complement

This manuscript describes two sites in a human IgG1 that, when mutated individually or in combination, result in a dramatic increase in C1q binding and complement-dependent cytotoxicity activity. These two residues, K326 and E333, are located at the extreme ends of the C1q binding epicenter in the CH2 domain of a human IgG. A mutation to tryptophan at K326 debilitates Ab-dependent cell-mediated cytotoxicity activity. In addition, substitutions of the residues E333 with serine and of K326 with tryptophan in a human IgG2 confer biological activity in the complement-dependent cytotoxicity assay in which the wild-type IgG2 is inactive. This study reveals that the residues K326 and E333 play a significant role in the control of the biological activity of an IgG molecule and can rescue the activity of an inactive IgG isotype.

Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer

The enhancement of immune effector functions has been proposed as a potential strategy for increasing the efficacy of therapeutic antibodies. Here, we show that removing fucose from trastuzumab (Herceptin) increased its binding to FcgammaRIIIa, enhanced antibody-dependent cell-mediated cytotoxicity, and more than doubled the median progression-free survival when compared with conventional trastuzumab in treating preclinical models of HER2-amplified breast cancer. Our results show that afucosylated trastuzumab has superior efficacy in treating in vivo models of HER2-amplified breast cancer and support the development of effector function-enhanced antibodies for solid tumor therapy.

Fas antigen signals proliferation of normal human diploid fibroblast and its mechanism is different from tumor necrosis factor receptor

Recent cloning of the cDNA for Fas/Apo‐1 and its ligand has revealed that they belong to the tumor necrosis factor (TNF) receptor and TNF family, respectively, and play an important role in apoptosis (programmed cell death). Like TNF, antibodies against the Fas antigen (anti‐Fas) have been shown to be cytotoxic to Fas‐expressing cells. Whether Fas, like TNF receptor, also mediates proliferation of normal human diploid fibroblasts (HDF), is not known. In this study, we show that HDF expresses Fas antigen and the engagement of this antigen signals proliferation of these cells in a dose‐dependent manner. Unlike TNF receptor, however, Fas‐mediated proliferation of HDF could not be blocked by orthovanadate, a tyrosine phosphatase inhibitor. The difference in the signaling was further evident from our observation that TNF induced the expression of interleukin‐6 but anti‐Fas did not. Overall, our results demonstrate for the first time that besides cell killing, Fas also mediates proliferation of HDF and that its mechanism is different from that of TNF receptor.

Erlotinib directly inhibits HER2 kinase activation and downstream signaling events in intact cells lacking epidermal growth factor receptor expression.

Erlotinib (Tarceva), is an orally available, reversible inhibitor of epidermal growth factor receptor (EGFR; HER1) that exhibits inhibitory activity on purified HER2 kinase at much higher concentrations. Despite the minimal activity on purified protein in vitro, in vivo studies show that erlotinib inhibits the growth of HER2-driven systems effectively. Several hypotheses have been put forward to explain this discrepancy. In particular, it has been suggested that erlotinib might indirectly suppress the activity of HER2 by blocking the ability of EGFR to transactivate it when the two receptors are part of a heterodimer complex. However, an alternative possibility that has not been adequately addressed is whether the direct inhibitory action of erlotinib on the HER2 kinase might account for the observed biological responses. To distinguish between a direct effect of erlotinib on HER2 kinase in intact cells or an indirect effect of erlotinib on HER2 activity that is mediated through EGFR, we generated cell lines that express either EGFR-H2 chimeric receptor or HER2 and HER3 receptors in an EGFR-negative background. We show that dose-dependent inhibition of HER2 was achieved at the receptor level, on downstream signaling molecules, and more importantly was also translated into inhibition of cell growth. Our findings imply that the inhibitory effect of erlotinib in HER2-expressing cells may in part be mediated through direct interaction with HER2 rather than indirectly through a process that requires the presence of EGFR.

Cooperation of the Agonistic DR5 Antibody Apomab with Chemotherapy to Inhibit Orthotopic Lung Tumor Growth and Improve Survival

Purpose: Apomab is a fully human monoclonal antibody that induces programmed cell death through the proapoptotic receptor DR5 in various cancer cells but not in normal cells. Several lung cancer cell lines express DR5 and exhibit apoptosis in response to apomab in vitro. Experimental Design: We investigated the efficacy of apomab and its interaction with chemotherapy in xenograft models based on human NCI-H460 non–small-cell lung carcinoma cells. In an established model of s.c. tumor xenografts, apomab or Taxol plus carboplatin chemotherapy delayed tumor progression, whereas combined treatment caused tumor regression and a substantially longer growth delay. To test apomab activity in a setting that may more closely mimic lung cancer pathology in patients, we developed a lung orthotopic model. Results: In this model, microcomputed tomography imaging showed that apomab, chemotherapy, or combination treatment significantly inhibited tumor growth compared with vehicle, whereas the combination caused greater inhibition in tumor growth relative to chemotherapy or apomab. Similarly, histologic analysis revealed that apomab, chemotherapy, or the combination significantly reduced tumor size compared with vehicle, whereas the combination induced significantly greater reduction in tumor size than did chemotherapy or apomab. Furthermore, combined treatment improved 105-day survival relative to vehicle (P = 0.0023) as well as to apomab (P = 0.0445) or chemotherapy (P = 0.0415). Conclusion: These results show a positive interaction of apomab with chemotherapy, evidenced by significant inhibition of tumor growth as well as improved survival, thus supporting further investigation of this therapeutic approach in lung cancer patients.

Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which both signal through IGF-I receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-II binding and by inducing cell surface receptor down-regulation via internalization and degradation, with the extracellular and intracellular domains of IGF-IR being differentially affected by the proteasomal and lysosomal inhibitors. In vitro, h10H5 exhibits antiproliferative effects on cancer cell lines. In vivo, h10H5 shows single-agent antitumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models and even greater efficacy in combination with the chemotherapeutic agent docetaxel or an anti–vascular endothelial growth factor antibody. Antitumor activity of h10H5 is associated with decreased AKT activation and glucose uptake and a 316-gene transcription profile with significant changes involving DNA metabolic and cell cycle machineries. These data support the clinical testing of h10H5 as a biotherapeutic for IGF-IR-dependent human tumors and furthermore illustrate a new method of monitoring its activity noninvasively in vivo via 2-fluoro-2-deoxy-d-glucose-positron emission tomography imaging. [Mol Cancer Ther 2008;7(9):2599–608]

Engineering upper hinge improves stability and effector function of a human IgG1.

Background: Radical reactions result in breakage of the heavy-light chain linkage and hinge cleavage of an IgG1. Results: The degraded products are generated by different reaction pathways and mechanisms. Conclusion: A His229/Tyr substitution improves stability and effector function of an IgG1. Significance: A mechanism based strategy to engineer the upper hinge to improve multiple properties of an IgG1 is feasible. Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys231 directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His229-mediated hinge cleavage. On the other hand, the substitution of His229 with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibodys binding to FcγRIII receptors by 2–3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed.

pp60ν-src kinase overexpression leads to cellular resistance to the antiproliferative effects of tumor necrosis factor

While some tumor cells are sensitive to the antiproliferative effects of tumor necrosis factor (TNF), others are resistant. The molecular basis for cellular resistance to TNF is not completely understood. Previously we have shown that transfection of cells with an oncogene HER2/neu/erb B2, a receptor tyrosine kinase, leads to resistance to the anticellular effects of TNF [(1988) Proc. Natl. Acad. Sci. USA 85, 5102‐5106]. In the present study, we demonstrate that the overexpression of another oncogenic tyrosine kinase, pp60 v‐src also induces resistance to TNF. In contrast to HER2, however, pp60 v‐src transfection of cells did not lead to down‐modulation of TNF receptors but rather to decreased intracellular glutathione levels. The pp60 v‐src ‐induced cellular resistance to TNF could be abrogated by interferon‐γ. Thus, these results indicate that the resistance of certain tumors to TNF may also be due in part to the overexpression of pp60 v‐src oncogene.

Transfection of cells with transforming growth factor‐α leads to cellular resistance to the antiproliferative effects of tumor necrosis factor

Tumor necrosis factor (TNF) is a growth‐modulatory cytokine that inhibits the growth of certain cell lines, stimulates the growth of some, and has no effect on the growth of still others. The molecular basis for this differential regulation of growth by TNF is not understood. We postulate that the growth of normal or tumor cells is determined by the balance between growth‐stimulatory and ‐inhibitory signals. In the present study, we demonstrate that the transfection of cells with the transforming growth factor (TGF)‐α gene induces resistance to TNF. Colon carcinoma cell lines that express elevated levels of TGF‐α were also found to be resistant to this cytokine. Exogenous addition of the growth factor was also effective in decreasing the antiproliferative effects of TNF. Transfection of cells with the TGF‐α gene led to downmodulation of TNF receptors but an increase in intracellular glutathione levels. Thus, these results support our hypothesis that expression of growth factors by certain tumor cells can lead to resistance to antiproliferative agents such as TNF.