Akiko Ishii-Watabe

Importance of Neonatal FcR in Regulating the Serum Half-Life of Therapeutic Proteins Containing the Fc Domain of Human IgG1: A Comparative Study of the Affinity of Monoclonal Antibodies and Fc-Fusion Proteins to Human Neonatal FcR

The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcγRI binding region of the Fc domain.

Optimization of transcriptional regulatory elements for constructing plasmid vectors

In studies regarding both gene therapy and gene function, transgene expression by plasmid vectors benefits from the use of transcriptional regulatory elements which permit high-level gene expression. Therefore, with respect to transgene (luciferase) expression activity both in vitro (using HeLa, HepG2, and ECV304 cells) and in vivo (mouse liver and skeletal muscle), we investigated the effective combination of commonly-used regulatory elements, such as the promoter/enhancer, intron, and polyadenylation signal (P(A)) sequence by constructing a series of plasmids that differed only in the particular sequence element being evaluated. Of the several promoter/enhancers that were tested, hybrid CA promoter/enhancer containing human cytomegalovirus immediate-early 1 gene (CMV) enhancer and chicken beta-actin promoter with the beta-actin intron sequence, and the improved CMV promoter/enhancer containing the largest intron of CMV (intron A) produced the highest levels of expression both in vitro and in vivo. P(A) sequences were found to have significant effects on transgene expression. The effect of a multiple enhancer was also examined. Optimized plasmids of this study were pCASL3 (composed of CMV enhancer, beta-actin promoter, beta-actin intron, Simian virus (SV40) P(A) sequence and SV40 enhancer) and pCMVSL3 (composed of CMV enhancer, CMV promoter, intron A, SV40 P(A) sequence and SV40 enhancer). These comparative analyses could provide a systematic reference for the development of vector construction for gene therapy, vaccine development, and gene transfer experiments.

CAR- or αv integrin-binding ablated adenovirus vectors, but not fiber-modified vectors containing RGD peptide, do not change the systemic gene transfer properties in mice

Targeted gene delivery to the tissue of interest by recombinant adenovirus (Ad) vectors is limited by the relatively broad expression of the primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, αv integrin. This problem could be overcome by mutating the fiber and penton base, which bind with CAR and αv integrin, respectively. In this study, we constructed CAR-binding ablated Ad vectors and αv integrin-binding ablated Ad vectors by mutation in the FG loop of fiber knob and in the RGD motif of penton base, respectively, and compared the gene transfer properties of their vectors into various types of cultured cells and mice with conventional Ad vectors. We also generated Ad vectors containing RGD peptide in the HI loop of the fiber knob. CAR-binding ablated Ad vectors mediated about 1% of gene transfer activity into CAR-positive cultured cells, compared with conventional Ad vectors, while αv integrin-binding ablated Ad vectors maintained at least 76% of gene transfer activity into cultured CAR-positive cells. Inclusion of the RGD peptide into the HI loop of the fiber knob of CAR-binding ablated Ad vectors restored gene transfer activity in vitro. On the other hand, systemically administered CAR-binding ablated Ad vectors, as well as αv integrin-binding ablated Ad vectors mediated similar levels of gene transfer into mouse liver with the conventional Ad vectors. These results suggest that continued interaction of either the fiber with CAR or the penton base with αv integrin offers an effective route of virus entry into mouse liver in vivo. Inhibition of the interaction of both the fiber with CAR and the penton base with αv integrin is likely to be crucial to the development of targeted Ad vectors.

Efficient gene transfer by fiber-mutant adenoviral vectors containing RGD peptide

One of the hurdles to adenovirus (Ad)-mediated gene transfer is that Ad vectors mediate inefficient gene transfer into cells lacking in the primary receptors, Coxsackievirus and adenovirus receptor (CAR). We previously developed a fiber-mutant Ad vector containing the Arg-Gly-Asp (RGD)-containing peptide motif on the HI loop of the fiber knob, and showed that the mutant vector had enhanced gene transfer activity to human glioma cells, which showed little CAR expression, compared to the vector containing wild type fiber. In this study, the feasibility of the Ad vector containing RGD peptide on the fiber knob was examined in a wide variety of cell types: CAR-positive or -negative human tumor cells, mouse cells, and leukemia cells. The mutant vector infected the cells, which lacked CAR expression but showed alpha(v) integrin expression, about 10-1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (alpha(v)beta3 and alpha(v)beta5)-dependent, CAR-independent cell entry pathway. The results of this study indicate that Ad vector containing RGD peptide on the fiber knob could be of great utility for gene therapy and gene transfer experiments.

Adenovirus Vector-Mediated Doxycycline-Inducible RNA Interference

RNA interference (RNAi) is a powerful tool for the knockdown of gene expression. Here, we report on the development of an adenovirus (Ad) vector-mediated doxycycline (Dox)-inducible small interfering RNA (siRNA) expression system. We used this siRNA system to control the expression of p53 and c-Myc in human cancer cells. Coinfection of Ad vectors containing the siRNA expression system under the control of the Dox-inducible H1 promoter and Ad vectors expressing a tetracycline repressor inhibited the expression levels of p53 and c-Myc in a dose-dependent manner with both Dox and viral dose. Regulated silencing of p53 and c-Myc expression was obtained. Because an Ad vector-mediated inducible RNAi system can efficiently transduce a variety of cell types in vitro and in vivo, and the degree of loss of gene expression can be modulated according to the dose of Dox, this expression system should be a useful tool for both basic research on the analysis of gene function and therapeutic applications of RNAi.

Strength evaluation of transcriptional regulatory elements for transgene expression by adenovirus vector

In studies of both gene function and gene therapy, transgene expression may be assisted considerably through the use of transcriptional regulatory elements with high activity. In this study, we evaluated the strength of various transcriptional regulatory elements both in vitro (six types of cell line) and in vivo (mouse heart, lung, kidney, spleen, and liver) by adenovirus-mediated gene transfer. In the case of the promoter/enhancer (P/E), the activity of CMV P/E (from the human cytomegalovirus immediate-early 1 gene) and hybrid CA P/E (composed of the CMV enhancer and chicken beta-actin promoter) were investigated, both of which are known to be strong and widely used. While hybrid CA P/E showed a higher transgene expression activity than CMV P/E, the addition of the intron A sequence (the largest intron of CMV) to CMV P/E increased the activity of CMV P/E to the same or higher level than that of hybrid CA P/E. Concerning the polyadenylation signal (P(A)) sequence, one from the bovine growth hormone (BGH) gene was about two times more efficient than that from the Simian virus 40 (SV40) late gene, both in vitro and in vivo. In the context of the CMV P/E containing the intron A sequence, a further increase in transgene expression was obtained by the addition of a SV40 enhancer downstream from the P(A) sequence. The combination of the SV40 P(A) and a SV40 enhancer showed almost comparable activity to BGH P(A). This information would be helpful for the construction of adenovirus vectors for studies regarding both gene function and gene therapy.

Development of a Cell-Based Assay Measuring the Activation of FcγRIIa for the Characterization of Therapeutic Monoclonal Antibodies

Antibody-dependent cellular cytotoxicity (ADCC) is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs). Among the human Fcγ receptors (FcγRs), FcγRIIIa is well known as the only receptor expressed in natural killer (NK) cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of FcγRIIa to mAb-mediated cytotoxicity have been reported. FcγRIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via FcγRIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human FcγRIIa-expressing reporter cell line. The FcγRIIa reporter cell assay was able to estimate the activation of FcγRIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate FcγRIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1) and panitumumab (IgG2). We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the FcγRIIa activation by EGFR-bound cetuximab. These results suggest that our FcγRIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.

Evaluation of intracellular trafficking and clearance from HeLa cells of doxorubicin-bound block copolymers.

New technologies are needed to deliver medicines safely and effectively. Polymeric nanoparticulate carriers are one such technology under investigation. We examined the intracellular trafficking of doxorubicin-bound block copolymers quantitatively and by imaging doxorubicin-derived fluorescence using confocal microscopy. The polymers were internalized by endocytosis and distributed in endosomal/lysosomal compartments and the endoplasmic reticulum; unlike free doxorubicin, the polymers were not found in the nucleus. Moreover, the ATP-binding cassette protein B1 (ABCB1) transporter may be involved in the efflux of the polymer from cells. This drug delivery system is attractive because the endogenous transport system is used for the uptake and delivery of the artificial drug carrier to the target as well as for its efflux from cells to medium. Our results show that a drug delivery system strategy targeting this endogenous transport pathway may be useful for affecting specific molecular targets.

Development of an Anti–Claudin-3 and -4 Bispecific Monoclonal Antibody for Cancer Diagnosis and Therapy

Most malignant tumors are derived from epithelium, and claudin (CLDN)-3 and CLDN-4 are frequently overexpressed in such tumors. Although antibodies have potential in cancer diagnostics and therapy, development of antibodies against CLDNs has been difficult because the extracellular domains of CLDNs are too small and there is high homology among human, rat, and mouse sequences. Here, we created a monoclonal antibody that recognizes human CLDN-3 and CLDN-4 by immunizing rats with a plasmid vector encoding human CLDN-4. A hybridoma clone that produced a rat monoclonal antibody recognizing both CLDN-3 and -4 (clone 5A5) was obtained from a hybridoma screen by using CLDN-3– and -4–expressing cells; 5A5 did not bind to CLDN-1–, -2–, -5–, -6–, -7–, or -9–expressing cells. Fluorescence-conjugated 5A5 injected into xenograft mice bearing human cancer MKN74 or LoVo cells could visualize the tumor cells. The human-rat chimeric IgG1 monoclonal antibody (xi5A5) activated FcγRIIIa in the presence of CLDN-3– or -4–expressing cells, indicating that xi5A5 may exert antibody-dependent cellular cytotoxicity. Administration of xi5A5 attenuated tumor growth in xenograft mice bearing MKN74 or LoVo cells. These results suggest that 5A5 shows promise in the development of a diagnostic and therapeutic antibody for cancers.

Discovery of anti-claudin-1 antibodies as candidate therapeutics against hepatitis C virus

Claudin-1 (CLDN1), a known host factor for hepatitis C virus (HCV) entry and cell-to-cell transmission, is a target molecule for inhibiting HCV infection. We previously developed four clones of mouse anti-CLDN1 monoclonal antibody (mAb) that prevented HCV infection in vitro. Two of these mAbs showed the highest antiviral activity. Here, we optimized the anti-CLDN1 mAbs as candidates for therapeutics by protein engineering. Although Fab fragments of the mAbs prevented in vitro HCV infection, their inhibitory effects were much weaker than those of the whole mAbs. In contrast, human chimeric IgG1 mAbs generated by grafting the variable domains of the mouse mAb light and heavy chains inhibited in vitro HCV infection as efficiently as the parental mouse mAbs. However, the chimeric IgG1 mAbs activated Fcγ receptor, suggesting that cytotoxicity against mAb-bound CLDN1-expressing cells occurred through the induction of antibody-dependent cellular cytotoxicity (ADCC). To avoid ADCC-induced side effects, we prepared human chimeric IgG4 mAbs. The chimeric IgG4 mAbs did not activate Fcγ receptor or induce ADCC, but they prevented in vitro HCV infection as efficiently as did the parental mouse mAbs. These findings indicate that the IgG4 form of human chimeric anti-CLDN1 mAb may be a candidate molecule for clinically applicable HCV therapy.