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Antibody interactions with Fcγ receptors (FcγRs), like FcγRIIIA, play a critical role in mediating antibody effector functions and thereby contribute significantly to the biologic and therapeutic activity of antibodies. Over the past decade, considerable work has been directed towards production of antibodies with altered binding affinity to FcγRs and evaluation of how the alterations modulate their therapeutic activity. This has been achieved by altering glycosylation status at N297 or by engineering modifications in the crystallizable fragment (Fc) region. While the effects of these modifications on biologic activity and efficacy have been examined, few studies have been conducted to understand their effect on antibody pharmacokinetics (PK). We present here a retrospective analysis in which we characterize the PK of three antibody variants with decreased FcγR binding affinity caused by amino acid substitutions in the Fc region (N297A, N297G, and L234A/L235A) and three antibody variants with increased FcγRIIIA binding affinity caused by afucosylation at N297, and compare their PK to corresponding wild type antibody PK in cynomolgus monkeys. For all antibodies, PK was examined at a dose that was known to be in the linear range. Since production of the N297A and N297G variants in Chinese hamster ovary cells results in aglycosylated antibodies that do not bind to FcγRs, we also examined the effect of expression of an aglycosylated antibody, without sequence change(s), in E. coli. All the variants demonstrated similar PK compared with that of the wild type antibodies, suggesting that, for the six antibodies presented here, altered FcγR binding affinity does not affect PK.

The phosphatidylinositol 3-kinase (PI3K) pathway is a central mediator of vascular endothelial growth factor (VEGF)-driven angiogenesis. The discovery of small molecule inhibitors that selectively target PI3K or PI3K and mammalian target of rapamycin (mTOR) provides an opportunity to pharmacologically determine the contribution of these key signaling nodes in VEGF-A-driven tumor angiogenesis in vivo. This study used an array of microvascular imaging techniques to monitor the antivascular effects of selective class I PI3K, mTOR, or dual PI3K/ mTOR inhibitors in colorectal and prostate cancer xenograft models. Micro-computed tomography (micro-CT) angiography, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), vessel size index (VSI) MRI, and DCE ultrasound (DCE-U/S) were employed to quantitatively evaluate the vascular (structural and physiological) response to these inhibitors. GDC-0980, a dual PI3K/mTOR inhibitor, was found to reduce micro-CT angiography vascular density, while VSI MRI demonstrated a significant reduction in vessel density and an increase in mean vessel size, consistent with a loss of small functional vessels and a substantial antivascular response. DCE-MRI showed that GDC-0980 produces a strong functional response by decreasing the vascular permeability/perfusion-related parameter, Ktrans. Interestingly, comparable antivascular effects were observed for both GDC-980 and GNE-490 (a selective class I PI3K inhibitor). In addition, mTOR-selective inhibitors did not affect vascular density, suggesting that PI3K inhibition is sufficient to generate structural changes, characteristic of a robust antivascular response. This study supports the use of noninvasive microvascular imaging techniques (DCE-MRI, VSI MRI, DCE-U/S) as pharmacodynamic assays to quantitatively measure the activity of PI3K and dual PI3K/mTOR inhibitors in vivo.

Granulocyte-colony stimulating factor (G-CSF) promotes mobilization of CD11b + Gr1 + myeloid cells and has been implicated in resistance to anti-VEGF therapy in mouse models. High G-CSF production has been associated with a poor prognosis in cancer patients. Here we show that activation of the RAS/MEK/ERK pathway regulates G-CSF expression through the Ets transcription factor. Several growth factors induced G-CSF expression by a MEK-dependent mechanism. Inhibition of G-CSF release with a MEK inhibitor markedly reduced G-CSF production in vitro and synergized with anti-VEGF antibodies to reduce CD11b + Ly6G + neutrophil mobilization and tumor growth and led to increased survival in animal models of cancer, including a genetically engineered mouse model of pancreatic adenocarcinoma. Analysis of biopsies from pancreatic cancer patients revealed increased phospho-MEK, G-CSF, and Ets expression and enhanced neutrophil recruitment compared with normal pancreata. These results provide insights into G-CSF regulation and on the mechanism of action of MEK inhibitors and point to unique anticancer strategies.

Purpose: We evaluated the prognostic and predictive use of circulating VEGF-A levels in phase III trials of bevacizumab in colorectal cancer, lung cancer, and renal cell carcinoma. Methods: Baseline plasma samples from 1,816 patients were analyzed for VEGF-A using an ELISA, which recognizes the major isoforms with equivalent sensitivity. HR and 95% confidence intervals (CI) for study end points were estimated using Cox regression analysis. A subset of matched archival tumor samples was analyzed for VEGF-A expression using in situ hybridization. Results: Higher VEGF-A levels showed trends toward adverse prognostic significance in the control arms of multiple trials, reaching statistical significance for overall survival (OS) in AVF2107 (highest vs. lowest 50%: HR = 1.76; 95% CI, 1.28–2.41), AVAiL (HR = 1.52; 95% CI, 1.16–2.00), and AVOREN (HR = 1.67; 95% CI, 1.18–2.36). In predictive analyses, the HRs for progression-free survival were similar across low and high VEGF-A subgroups and favored bevacizumab-containing treatment. In the low VEGF-A subgroups, HRs (95% CIs) were 0.61 (0.43–0.87) in AVF2107, 0.71 (0.43–1.16) in E4599, 0.74 (0.59–0.94) in AVAiL (low-dose), 0.89 (0.70–1.13) in AVAiL (high-dose), and 0.56 (0.40–0.78) in AVOREN. Analyses of OS data have shown similar results. No correlation between primary tumor VEGF-A expression and plasma VEGF-A levels was observed. Conclusions: In this comprehensive evaluation, pretreatment total circulating VEGF-A was prognostic for outcome in metastatic colorectal, lung, and renal cell cancers, but it was not predictive for bevacizumab-based treatment benefit. Clin Cancer Res; 19(4); 929–37. ©2012 AACR.

Retailers are increasingly utilizing self-service technology (SST) in the delivery of services to their consumers. This study confirms prior findings that the SST attributes of perceived usefulness, ease of use, reliability, and fun all have a direct influence on consumers' attitude toward using SST to complete retail transactions. This study also expands earlier research by investigating the impact technology readiness (TR) has on consumers' perceived reliability and perceived fun of using SST. Implications are provided to help retailers manage consumers' perceptions and expectations related to using SST to complete retail transactions, as well as strategies to enhance consumer technology competence.

This article contributes to scholarly understanding of the significance of procedural fairness in pricing contexts. It has been widely recognized that price fairness judgments concern both the outcome (fair price) and the procedure leading to the outcome (fair pricing). However, extant research has traditionally viewed procedural fairness as a means to outcome fairness. According to this instrumental view, procedural fairness is a component or antecedent of outcome fairness, but has no direct effects on consumers’ responses to prices. Building on the relational perspective on fairness, we develop and test a model of price procedural fairness as an end in itself. In three lab studies, we show that (1) when information regarding outcome (an unfavorable price difference) and procedure (the pricing practice underlying the price difference) is available simultaneously and unambiguously, procedural fairness has direct and stronger effects than outcome fairness on consumers’ responses and (2) procedural fairness mediates the effects of pricing practices on these responses. In all three studies, adding procedural fairness as a direct predictor of consumers’ responses increases the explanatory power of a model of price fairness significantly. Our model can explain peculiar real-world cases in which consumers reacted very strongly over relatively small price differences. The research findings point to the significance of the non-instrumental aspect of consumer’s demand for ethical (fair pricing) behavior and the need for companies to assess the fairness of their pricing practices from the consumer perspective.

Bv8, also known as prokineticin 2, has been characterized as an important mediator of myeloid cell mobilization and myeloid cell-dependent tumor angiogenesis. Bv8 expression is dramatically enhanced by G-CSF, both in vitro and in vivo. The mechanisms involved in such up-regulation remain unknown. Using pharmacological inhibitors that interfere with multiple signaling pathways known to be activated by G-CSF, we show that signal transducer and activator of transcription 3 (Stat3) activation is required for Bv8 up-regulation in mouse bone marrow cells, whereas other Stat family members and extracellular signal-regulated kinase (ERK) activation are not involved. We further identified CD11b+ Gr1+ myeloid cells as the primary cell population in which Stat3 signaling is activated by G-CSF. Bv8 expression induced by G-CSF was also significantly reduced by siRNA-mediated Stat3 knockdown. Moreover, chromatin immunoprecipitation studies indicate that G-CSF significantly induces binding of phospho-Stat3 to the Bv8 promoter, which was abolished by pretreatment with the Stat3 inhibitor WP1066. Luciferase assay confirmed that the phospho-Stat3 binding site is a functional enhancer of the Bv8 promoter. The key role of Stat3 signaling in regulating G-CSF-induced Bv8 expression was further confirmed by in vivo studies. We show that the regulation of Bv8 expression in human bone marrow cells is also Stat3 signaling-dependent. Stat3 is recognized as a key regulator of inflammation-dependent tumorigenesis. We propose that such a role of Stat3 reflects at least in part its ability to regulate Bv8 expression.

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 Cys(231) directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His(229)-mediated hinge cleavage. On the other hand, the substitution of His(229) 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 antibody's 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.

The neonatal Fc receptor (FcRn) plays an important and well-known role in immunoglobulin G (IgG) catabolism; however, its role in the disposition of IgG after subcutaneous (SC) administration, including bioavailability, is relatively unknown. To examine the potential effect of FcRn on IgG SC bioavailability, we engineered three anti-amyloid β monoclonal antibody (mAb) reverse chimeric mouse IgG2a (mIgG2a) Fc variants (I253A.H435A, N434H and N434Y) with different binding affinities to mouse FcRn (mFcRn) and compared their SC bioavailability to that of the wild-type (WT) mAb in mice. Our results indicated that the SC bioavailability of mIgG2a was affected by mFcRn-binding affinity. Variant I253A.H435A, which did not bind to mFcRn at either pH 6.0 or pH 7.4, had the lowest bioavailability (41.8%). Variant N434Y, which had the greatest increase in binding affinity at both pH 6.0 and pH 7.4, had comparable bioavailability to the WT antibody (86.1% vs. 76.3%), whereas Variant N434H, which had modestly increased binding affinity at pH 6.0 to mFcRn and affinity comparable to the WT antibody at pH 7.4, had the highest bioavailability (94.7%). A semi-mechanism-based pharmacokinetic model, which described well the observed data with the WT antibody and variant I253A.H435A, is consistent with the hypothesis that the decreased bioavailability of variant I253A.H435A was due to loss of the FcRn-mediated protection from catabolism at the absorption site. Together, these data demonstrate that FcRn plays an important role in SC bioavailability of therapeutic IgG antibodies.

Internet-based retailing is growing and increasingly becoming popular as an alternative to traditional retailing around the world. Increased research interest in the quality of e-retailer service has led to the development of domain-specific models. The new models, however, have not been extensively tested, and their generalizability remains to be established. The present study undertakes the task of extending the available models by incorporating the influence of cultural factors and empirically testing the proposed model. Data from China and the United States are employed for this purpose. The results provide partial support for the proposed model.

Summary Limitations to the application of molecularly targeted cancer therapies are the inability to accurately match patient with effective treatment and the absence of a prompt readout of posttreatment response. Noninvasive agents that rapidly report vascular endothelial growth factor (VEGF) levels using positron emission tomography (PET) have the potential to enhance anti-angiogenesis therapies. Using phage display, two distinct classes of peptides were identified that bind to VEGF with nanomolar affinity and high selectivity. Co-crystal structures of these different peptide classes demonstrate that both bind to the receptor-binding region of VEGF. 18 F-radiolabelling of these peptides facilitated the acquisition of PET images of tumor VEGF levels in a HM7 xenograph model. The images obtained from one 59-residue probe, 18 F-Z-3B, 2 hr postinjection are comparable to those obtained with anti-VEGF antibody B20 72 hr postinjection. Furthermore, VEGF levels in growing SKOV3 tumors were followed using 18 F-Z-3B as a PET probe with VEGF levels increasing with tumor size.

SummaryAntibodies to cell-surface antigens trigger activatory Fcγ receptor (FcγR)-mediated retrograde signals in leukocytes to control immune effector functions. Here, we uncover an FcγR mechanism that drives antibody-dependent forward signaling in target cells. Agonistic antibodies to death receptor 5 (DR5) induce cancer-cell apoptosis and are in clinical trials; however, their mechanism of action in vivo is not fully defined. Interaction of the DR5-agonistic antibody drozitumab with leukocyte FcγRs promoted DR5-mediated tumor-cell apoptosis. Whereas the anti-CD20 antibody rituximab required activatory FcγRs for tumoricidal function, drozitumab was effective in the context of either activatory or inhibitory FcγRs. A CD40-agonistic antibody required similar FcγR interactions to stimulate nuclear factor-κB activity in B cells. Thus, FcγRs can drive antibody-mediated receptor signaling in target cells.

In this study, we reviewed the quality of customer service (E-S-QUAL) model and the quality of service recovery (E-RecS-QUAL) model and applied them to the African American and Chinese cultural settings. Utilizing the LISREL 8.72 program, we conducted measurement equivalence tests and found that the e-service quality measurement can be generalized to different cultures, and the equivalence of measurement between two cultures considered here is partially supported. In addition, t-tests were conducted on each dimension of Web service quality in order to compare the mean differences between the perceptions of Chinese and African American consumers. It was found that Chinese consumers rate Web service quality at a significantly lower level than African American consumers on all seven dimensions. Methodological and practical implications of the study are discussed.

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 FcγRIIIa, 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. Cancer Res; 70(11); 4481–9. ©2010 AACR.

IgE-mediated hypersensitivity is central to the pathogenesis of asthma and other allergic diseases. Although neutralization of serum IgE with IgE-specific antibodies is in general an efficacious treatment for allergic asthma, one limitation of this approach is its lack of effect on IgE production. Here, we have developed a strategy to disrupt IgE production by generating monoclonal antibodies that target a segment of membrane IgE on human IgE-switched B cells that is not present in serum IgE. This segment is known as the M1′ domain, and using genetically modified mice that contain the human M1′ domain inserted into the mouse IgE locus, we demonstrated that M1′-specific antibodies reduced serum IgE and IgE-producing plasma cells in vivo, without affecting other immunoglobulin isotypes. M1′-specific antibodies were effective when delivered prophylactically and therapeutically in mouse models of immunization, allergic asthma, and Nippostrongylus brasiliensis infection, likely by inducing apoptosis of IgE-producing B cells. In addition, we generated a humanized M1′-specific antibody that was active on primary human cells in vivo, as determined by its reduction of serum IgE levels and IgE plasma cell numbers in a human PBMC-SCID mouse model. Thus, targeting of human IgE-producing B cells with apoptosis-inducing M1′-specific antibodies may be a novel treatment for asthma and allergy.

Generalizability of measurement instruments across different cultures is becoming more important as marketers increasingly engage in cross-cultural research. It is essential to assess whether instruments used to measure relevant constructs in one culture can also be applied to other cultures before any cultural comparisons can be made. The findings of this study suggest that the Technology Readiness Index (Parasuraman 2000) is a cross-culturally valid measurement scale for both American and Chinese consumers. The same four technology-readiness dimensions exist for both consumer groups. This study also provides additional insights into the methodological foundation for cross-cultural studies.

PurposeThe purpose of this paper is to apply Lichtenstein et al.'s price perception model to American, Chinese and Japanese cultures, to test the measurement equivalence across three cultures, and to compare the price perception constructs across three cultures using equivalent instruments.Design/methodology/approachA questionnaire is used to collect information on more than 500 student respondents from America, China, and Japan.FindingsUtilizing structural equation modeling, a 21‐item version of Lichtenstein et al.'s scale is created that has good fit across the three cultures. In progressively constraining tests, good model fit is found when constraining or partially constraining the factor loadings, error correlations, factor variances, and correlations between factors to be equal across three cultures tested. In addition, after creating price perception subscales, no significant differences emerge between Chinese, Japanese, and US consumers on value consciousness or price/quality schemas. Significant differences emerge on price consciousness, prestige sensitivity, and sales proneness.Practical implicationsThe 21‐item scale of Lichtenstein et al.'s price perception model can be generalized to both China and Japan. The primary conclusions (i.e. that Chinese consumers reported significantly higher price and prestige sensitivity, compared to USA and Japanese consumers, while US consumers showed higher levels of sales proneness than Chinese and Japanese consumers) provide a rationale for international retailers to develop different pricing and promotional strategies when expanding their business into these three cultures.Originality/valueA 21‐item scale to measure five of Lichtenstein et al.'s price perception constructs that has been validated through measurement invariance tests and compared across consumers in China, Japan, and the USA is provided.

Neuropilin-1 (NRP1) acts as a co-receptor for class 3 semaphorins and vascular endothelial growth factor and is an attractive angiogenesis target for cancer therapy. In addition to the transmembrane form, naturally occurring soluble NRP1 proteins containing part of the extracellular domain have been identified in tissues and a cell line. We developed ELISAs to study the existence of circulating NRP1 and to quantify it in serum. As measured by ELISAs, circulating NRP1 levels in mice, rats, monkeys and humans were 427 +/- 77, 20 +/- 3, 288 +/- 86 and 322 +/- 82 ng/ml (mean +/- standard deviation; n > or = 10), respectively. Anti-NRP1(B), a human monoclonal antibody, has been selected from a synthetic phage library. A 4-fold increase in circulating NRP1 was observed in mice receiving a single dose of 10 mg/kg anti-NRP1(B) antibody. In rats and monkeys receiving single injections of anti-NRP1(B) at different dose levels, higher doses of antibody resulted in greater and more prolonged increases in circulating NRP1. Maximum increases were 56- and 7-fold for rats and monkeys receiving 50 mg/kg anti-NRP1(B), respectively. In addition to the soluble NRP1 isoforms, for the first time, a approximately 120 kDa circulating NRP1 protein containing the complete extracellular domain was detected in serum by western blot and mass spectrometry analysis. This protein increased more than the putative soluble NRP1 bands in anti-NRP1(B) treated mouse, rat and monkey sera compared with untreated controls, suggesting that anti-NRP1(B) induced membrane NRP1 shedding.

Purpose – This paper seeks to use the consumer‐perceived levels of internet shopping skills and challenges, to cluster the young Chinese customers and to compare the quality perceptions of customers from the different clusters.Design/methodology/approach – A survey of 237 college students from Beijing with the average age of 20.2 was conducted via a paper‐and‐pencil questionnaire. All the constructs were measured using established scales. The students completed the questionnaire in their native language.Findings – The paper finds that, at the present level of internet development in China, online consumers can be segmented on the basis of their self‐rated internet skills and their perception of the challenges involved in online shopping.Practical implications – Online customer segments obtained through internet skill and challenge level perceptions are demonstrated to explain some of the differences in the online shopping behaviors and service quality perceptions. The managers of online stores in China co...

The development of effective retailing strategies that are sensitive to cross-cultural differences would seem to be of considerable importance to their success in the global marketplace. Building on two existing models, SERVQUAL and RSQS, this study developed scales to examine service quality in Hong Kong's supermarkets. Based on intensive field study, we revised the existing service quality instruments and developed a new set of instruments to measure service quality in Hong Kong's markets. A new underlying structure emerged, suggesting that in Hong Kong, Chinese consumers perceived service quality in the regular supermarkets based on their purchasing process instead of from the tangible and non-tangible aspects found in previous studies. In the enhanced supermarkets, however, consumers perceived service quality differently than did the consumers in the regular type of store, suggesting a different model would be appropriate. Overall study results indicated that the measurement and underlying structure of service quality perception was not only industry and culture specific, but also specific to the form of retail structures that may enter the cultural mélange of the Chinese marketplace.

Relationship quality is increasingly emerging as a strategy for organizations that strive to retain loyal and satisfied customers in today's highly competitive environment. However, a limited number of studies have investigated relationship quality within the hospitality industry. This study examines and applies a measurement model originally tested with Korean customers to American customers to confirm that the predictors of relationship quality for luxury restaurants are cross-culturally valid. This study also examines the relative importance of each predictor of relationship quality, and identifies strategies for luxury restaurants that should enhance their level of customer trust and satisfaction.

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.

The creation of an effective learning environment requires cultural competency – the ability to interact effectively with people of different cultures. Cultural competency means knowing and understanding the people that you serve. This study compares American and Chinese student’s readiness and willingness to use innovative technology by assessing their technology readiness through the use of the Technology Readiness Index (Parasuraman, 2000). The findings show that Chinese students exhibit higher levels of discomfort and insecurity, and lower levels of optimism and innovativeness with regard to using new technology. Implications for cross-cultural technology-based learning environments are also provided.

In this study, we applied a measurement model of the country-of-origin (CO) effect developed by Parameswaran and Pisharodi (1994) across three groups: Caucasian Americans, African-Americans, and Chinese consumers. We explored CO effects towards a tangible product with a different origin country (Japan) than previously examined and explored the CO effect towards a service offering. Utilizing structural equation modeling, we found that although the facets of CO effect developed by other authors were robust in different consumer purchase settings, the items measuring each factor showed significant discrepancy. Both theoretical and managerial contributions are discussed and future research is suggested.

Thymic stromal lymphopoietin (TSLP) potently induces deregulation of Th2 responses, a hallmark feature of allergic inflammatory diseases such as asthma, atopic dermatitis, and allergic rhinitis. However, direct downstream in vivo mediators in the TSLP-induced atopic immune cascade have not been identified. In our current study, we have shown that OX40 ligand (OX40L) is a critical in vivo mediator of TSLP-mediated Th2 responses. Treating mice with OX40L-blocking antibodies substantially inhibited immune responses induced by TSLP in the lung and skin, including Th2 inflammatory cell infiltration, cytokine secretion, and IgE production. OX40L-blocking antibodies also inhibited antigen-driven Th2 inflammation in mouse and nonhuman primate models of asthma. This treatment resulted in both blockade of the OX40-OX40L receptor-ligand interaction and depletion of OX40L-positive cells. The use of a blocking, OX40L-specific mAb thus presents a promising strategy for the treatment of allergic diseases associated with pathologic Th2 immune responses.

Neuropilin-1 (NRP1) is a single spanning transmembrane glycoprotein that acts as a co-receptor for class 3 semaphorins and vascular endothelial growth factors. Naturally occurring soluble NRP1 isoforms containing partial extracellular domain (ECD) have been reported. In addition to soluble NRP1, full-length NRP1 ECD has also been identified in human and animal sera. Here, we describe primate and rodent NRP1 ELISAs that measure total circulating NRP1 including soluble NPR1 and NRP1 ECD in human, monkey, mouse, and rat sera.

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and plays an important role in physiological and tumor angiogenesis. The human VEGF gene has eight exons. Different VEGF isoforms are expressed via alternative RNA splicing and VEGF121 and VEGF165 are the major isoforms present in human tissues. The exact roles of these different VEGF isoforms are not totally clear. Assays to detect specific VEGF isoforms in biological samples are needed to understand the biological functions of these different VEGF isoforms and to better assess their potential use as predicative biomarkers for anti-angiogenic therapy. Because monoclonal antibodies specific to different VEGF isoforms are lacking, we used antibodies directed to different epitopes on VEGF165 in a set of three enzyme-linked immunosorbent assays (ELISAs) to assess the amount of VEGF121 and VEGF165 as well as VEGF110, which can be generated by plasmin cleavage in vivo. The first ELISA detects VEGF165. The second ELISA detects both VEGF121 and VEGF165. The third ELISA detects VEGF165, VEGF121, and VEGF110. The concentrations of VEGF121 can be assessed from the difference in VEGF concentrations measured by the second and the first ELISAs; the concentrations of VEGF110 can be assessed from the difference in VEGF concentrations measured by the third and the second ELISAs. The same assay strategy may be used to assess the amount of other VEGF isoforms if antibodies directed against the desired amino acids in those isoforms can be obtained.

Neuropilin-1 (NRP1) is a single spanning transmembrane glycoprotein that acts as a co-receptor for class 3 semaphorins and vascular endothelial growth factors. Naturally occurring soluble NRP1 isoforms containing partial extracellular domain (ECD) have been reported. In addition to soluble NRP1, full-length NRP1 ECD has also been identified in human and animal sera. Here, we describe primate and rodent NRP1 ELISAs that measure total circulating NRP1 including soluble NPR1 and NRP1 ECD in human, monkey, mouse, and rat sera.

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and plays an important role in physiological and tumor angiogenesis. The human VEGF gene has eight exons. Different VEGF isoforms are expressed via alternative RNA splicing and VEGF121 and VEGF165 are the major isoforms present in human tissues. The exact roles of these different VEGF isoforms are not totally clear. Assays to detect specific VEGF isoforms in biological samples are needed to understand the biological functions of these different VEGF isoforms and to better assess their potential use as predicative biomarkers for anti-angiogenic therapy. Because monoclonal antibodies specific to different VEGF isoforms are lacking, we used antibodies directed to different epitopes on VEGF165 in a set of three enzyme-linked immunosorbent assays (ELISAs) to assess the amount of VEGF121 and VEGF165 as well as VEGF110, which can be generated by plasmin cleavage in vivo. The first ELISA detects VEGF165. The second ELISA detects both VEGF121 and VEGF165. The third ELISA detects VEGF165, VEGF121, and VEGF110. The concentrations of VEGF121 can be assessed from the difference in VEGF concentrations measured by the second and the first ELISAs; the concentrations of VEGF110 can be assessed from the difference in VEGF concentrations measured by the third and the second ELISAs. The same assay strategy may be used to assess the amount of other VEGF isoforms if antibodies directed against the desired amino acids in those isoforms can be obtained.

The MHC class I-like Fc receptor FcRn plays an essential role in extending the half-life (t(1/2)) of IgG antibodies and IgG-Fc-based therapeutics in the circulation. The goal of this study was to analyze the effect of human IgG1 (hIgG1) antibodies with enhanced in vitro binding to FcRn on their in vivo t(1/2) in mice expressing human FcRn (hFcRn). Mutants of the humanized monoclonal Herceptin antibody (Hu4D5-IgG1), directed against human epidermal growth factor receptor 2 (p185 (HER2)), show altered pH-dependent binding to hFcRn in vitro. Two engineered IgG1 mutants (N434A and T307A/E380A/N434A) showed a considerably extended t(1/2) in vivo compared with wild-type antibody in mice expressing an hFcRn transgene (Tg) but not in mice expressing the endogenous mouse FcRn. The efficiency of hFcRn-mediated protection was dependent on hFcRn Tg copy number. Moreover, when injected into FcRn-humanized mice at a concentration sufficient to partially saturate hFcRn, the engineered IgG1 mutants with an extended serum t(1/2) were most effective in reducing the t(1/2) of a tracer hIgG1 antibody. Finally, administration of mutant with high binding to hFcRn ameliorated arthritis induced by passive transfer with human pathogenic plasma. These results indicate that Fc regions modified for high binding affinity to hFcRn increases serum persistence of therapeutic antibodies, that the same approach can be exploited as an anti-autoimmune therapy to promote the clearance of endogenous pathogenic IgG and that FcRn-humanized mice are a promising surrogate for hIgG therapeutic development.

A cell-based ELISA using suspension WIL2 cells in 96-well format was previously developed for measuring relative binding affinities of humanized anti-CD20 variants. We further developed a new cell-binding assay that uses high binding capacity carbon electrode plates for rapid attachment of suspension WIL2 cells and electrochemiluminescence for detection. Compared to the cell-based ELISA, which requires centrifugation for the manual wash steps, significant improvement in assay throughput was achieved by using a microplate washer. The assay can be performed on both 96- and 384-well plates with a standard curve range of 2.74-2000 ng/ml, which is wider than the range of 15.6-1000 ng/ml for the cell-based ELISA. Using CD20 expressing CHO cell clones, surface expression of >or=33,000 CD20 molecules was sufficient to obtain a dose-response curve in 384-well format. Relative affinities of 15 humanized variants correlated well (r(2)=0.94) between electrochemiluminescent cell-binding assay and cell-based ELISA. A competitive assay format, using mouse anti-CD20 antibody as the tracer, with a dose-response range of 27.4-20,000 ng/ml was also developed. The new cell-binding assay method can be used to efficiently support humanization process for selection of anti-CD20 antibody drug candidates and to characterize antibody binding to other cell surface proteins.

Activated fibroblasts are thought to play important roles in the progression of many solid tumors, but little is known about the mechanisms responsible for the recruitment of fibroblasts in tumors. Using several methods, we identified platelet-derived growth factor A (PDGFA) as the major fibroblast chemoattractant and mitogen from conditioned medium generated by the Calu-6 lung carcinoma cell line. In addition, we showed that Calu-6 tumors express significant levels of PDGFC, and that the levels of expression of these two PDGFRα ligands correlate strongly with the degree of stromal fibroblast infiltration into the tumor mass. The most intense expression of PDGFRα was observed in fibroblasts in the tumor outer rim. We subsequently showed that disrupting PDGFRα-mediated signaling results in significant inhibition of tumor growth in vivo. Furthermore, analysis of a compendium of microarray data revealed significant expression of PDGFA, PDGFC, and PDGFRα in human lung tumors. We propose that therapies targeting this stromal cell type may be effective in treating certain types of solid tumors.

Vascular endothelial growth factor-A (VEGF-A) and its 2 transmembrane tyrosine-kinase receptors, VEGFR-1 and VEGFR-2, constitute a ligand-receptor signaling system that is crucial for developmental angiogenesis. VEGF-B and placental growth factor (PlGF) activate VEGFR-1 selectively, however, mice lacking either ligand display only minor developmental defects. We hypothesized that the relative contributions of VEGF-B and PlGF to VEGFR-1 signaling may be masked in the presence of VEGF-A, which is abundantly expressed during postnatal development. To test this hypothesis, neonatal or adult mice were treated with a monoclonal antibody (G6-23-IgG) blocking murine VEGF-A or a soluble VEGFR-1 receptor IgG chimeric construct [mFlt(1-3)-IgG], which neutralizes VEGF-A, VEGF-B, and PlGF. Both compounds attenuated growth and survival of neonatal mice to similar extents and the pathophysiologic alterations, including a reduction in organ size and vascularization, changes in gene expression, and hematologic end points, were essentially indistinguishable. In adult mice, we observed only minor changes in response to treatment, which were similar between both anti-VEGF compounds. In conclusion, our findings suggest that PlGF and VEGF-B do not compensate during conditions of VEGF-A blockade, suggesting a minor role for compensatory VEGFR-1 signaling during postnatal development and vascular homeostasis in adults. The absence of compensatory VEGFR-1 signaling by VEGF-B and PlGF may have important implications for the development of anticancer strategies targeting the VEGF ligand/receptor system.

B cell immunotherapy has emerged as a mainstay in the treatment of lymphomas and autoimmune diseases. Although the microenvironment has recently been demonstrated to play critical roles in B cell homeostasis, its contribution to immunotherapy is unknown. To analyze the in vivo factors that regulate mechanisms involved in B cell immunotherapy, we used a murine model for human CD20 (hCD20) expression in which treatment of hCD20+ mice with anti-hCD20 mAbs mimics B cell depletion observed in humans. We demonstrate in this study that factors derived from the microenvironment, including signals from the B cell-activating factor belonging to the TNF family/BLyS survival factor, integrin-regulated homeostasis, and circulatory dynamics of B cells define distinct in vivo mechanism(s) and sensitivities of cells in anti-hCD20 mAb-directed therapies. These findings provide new insights into the mechanisms of immunotherapy and define new opportunities in the treatment of cancers and autoimmune diseases.

Background— The mechanisms underlying the variation in collateral formation between patients, even with similar patterns of coronary artery disease, remain unclear. This study investigates whether circulating humoral or cellular factors can provide an insight into this variation. Methods and Results— Thirty patients with isolated left anterior descending coronary artery disease underwent percutaneous coronary intervention with collateral flow index (CFI) determined using a pressure wire. Patients with inadequate (CFI r =−0.61, P 165 , or placental growth factor. There was a strong positive correlation between numbers of CD34/CD133-positive circulating hemopoietic precursor cells and CFI ( r =0.75, P P P Conclusions— In this study, inadequate coronary collateral development is associated with reduced numbers of circulating EPCs and impaired chemotactic and proangiogenic but not mitogenic activity. These findings are consistent with current efforts to enhance collateral formation by augmentation of circulating EPCs.

To evaluate the toxicity and activity of thalidomide in patients with advanced metastatic renal cell cancer and to measure changes of one angiogenic factor, vascular endothelial growth factor (VEGF)165, with therapy.29 patients were enrolled on a study of thalidomide using an intra-patient dose escalation schedule. Patients began thalidomide at 400 mg/d and escalated as tolerated to 1200 mg/d by day 54. Fifty-nine per cent of patients had had previous therapy with IL-2 and 52% were performance status 2 or 3. Systemic plasma VEGF165 levels were measured by dual monoclonal ELISA in 8 patients.24 patients were evaluable for response with one partial response of 11 months duration of a patient with hepatic and pulmonary metastases (4%), one minor response, and 2 patients stable for over 6 months. Somnolence and constipation were prominent toxicities and most patients could not tolerate the 1200 mg/day dose level. Systemic plasma VEGF165 levels did not change with therapy.These results are consistent with a low level of activity of thalidomide in renal cell carcinoma. Administration of doses over 800 mg/day was difficult to achieve in this patient population, however lower doses were practical. The dose-response relationship, if any, of thalidomide for renal cell carcinoma is unclear.

Plasma protein binding can be an effective means of improving the pharmacokinetic properties of otherwise short lived molecules. Using peptide phage display, we identified a series of peptides having the core sequence DICLPRWGCLW that specifically bind serum albumin from multiple species with high affinity. These peptides bind to albumin with 1:1 stoichiometry at a site distinct from known small molecule binding sites. Using surface plasmon resonance, the dissociation equilibrium constant of peptide SA21 (Ac-RLIEDICLPRWGCLWEDD-NH(2)) was determined to be 266 +/- 8, 320 +/- 22, and 467 +/- 47 nm for rat, rabbit, and human albumin, respectively. SA21 has an unusually long half-life of 2.3 h when injected by intravenous bolus into rabbits. A related sequence, fused to the anti-tissue factor Fab of D3H44 (Presta, L., Sims, P., Meng, Y. G., Moran, P., Bullens, S., Bunting, S., Schoenfeld, J., Lowe, D., Lai, J., Rancatore, P., Iverson, M., Lim, A., Chisholm, V., Kelley, R. F., Riederer, M., and Kirchhofer, D. (2001) Thromb. Haemost. 85, 379-389), enabled the Fab to bind albumin with similar affinity to that of SA21 while retaining the ability of the Fab to bind tissue factor. This interaction with albumin resulted in reduced in vivo clearance of 25- and 58-fold in mice and rabbits, respectively, when compared with the wild-type D3H44 Fab. The half-life was extended 37-fold to 32.4 h in rabbits and 26-fold to 10.4 h in mice, achieving 25-43% of the albumin half-life in these animals. These half-lives exceed those of a Fab'(2) and are comparable with those seen for polyethylene glycol-conjugated Fab molecules, immunoadhesins, and albumin fusions, suggesting a novel and generic method for improving the pharmacokinetic properties of rapidly cleared proteins.

Vascular endothelial growth factor (VEGF) is a principal regulator of blood vessel formation and haematopoiesis, but the mechanisms by which VEGF differentially regulates these processes have been elusive. Here we describe a regulatory loop by which VEGF controls survival of haematopoietic stem cells (HSCs). We observed a reduction in survival, colony formation and in vivo repopulation rates of HSCs after ablation of the VEGF gene in mice. Intracellularly acting small-molecule inhibitors of VEGF receptor (VEGFR) tyrosine kinase dramatically reduced colony formation of HSCs, thus mimicking deletion of the VEGF gene. However, blocking VEGF by administering a soluble VEGFR-1, which acts extracellularly, induced only minor effects. These findings support the involvement in HSC survival of a VEGF-dependent internal autocrine loop mechanism (that is, the mechanism is resistant to inhibitors that fail to penetrate the intracellular compartment). Not only ligands selective for VEGF and VEGFR-2 but also VEGFR-1 agonists rescued survival and repopulation of VEGF-deficient HSCs, revealing a function for VEGFR-1 signalling during haematopoiesis.

Pharmacokinetic (PK) testing of a humanized (κI, VH3 framework) and affinity matured anti-hepatitis C virus E2-glycoprotein (HCV-E2) antibody (hu5B3.κ1VH3.v3) in rats revealed unexpected fast clearance (34.9 mL/day/kg). This antibody binds to the rat recycling receptor FcRn as expected for a human IgG1 antibody and does not display non-specific binding to baculovirus particles in an assay that is correlated with fast clearance in cynomolgus monkey. The antigen is not expressed in rat so target-dependent clearance does not contribute to PK. Removal of the affinity maturation changes (hu5B3.κ1VH3.v1) did not restore normal clearance. The antibody was re-humanized on a κ4, VH1 framework and the non-affinity matured version (hu5B3.κ4VH1.v1) was shown to have normal clearance (8.5 mL/day/kg). Since the change in framework results in a lower pI, primarily due to more negative charge on the κ4 template, the effect of additional charge variation on antibody PK was tested by incorporating substitutions obtained through phage display affinity maturation of hu5B3.κ1VH3.v1. A variant having a pI of 8.61 gave very fast clearance (140 mL/day/kg) whereas a molecule with pI of 6.10 gave slow clearance (5.8 mL/kg/day). Both antibodies exhibited comparable binding to rat FcRn, but biodistribution experiments showed that the high pI variant was catabolized in liver and spleen. These results suggest antibody charge can have an effect on PK through alterations in antibody catabolism independent of FcRn-mediated recycling. Furthermore, introduction of affinity maturation changes into the lower pI framework yielded a candidate with PK and virus neutralization properties suitable for clinical development.

In March, 2005, Japan attempted to bid for a permanent seat on the United Nations (UN) Security Council. Millions of Chinese protested strongly against it because Japan’s invasion and partial occupation of China in World War II. Demonstrations opposing Japan’s UN bid were conducted in almost every province in China. The voices of anti-Japan, anti-Japanese product, and even boycotting Japanese products rose higher and higher.

Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2–1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective [1]–[3]. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans., Author Summary Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection and causes developmental abnormalities, including hearing loss and developmental delay. Although there is no therapy for congenital HCMV disease, there is evidence from both human and animal studies that antibodies can have efficacy in this setting. Such studies have focused exclusively on polyclonal antibodies, in which the targets of protective antibodies are unknown. Guinea pigs have been used as a model of human maternal fetal transmission of infection because of similarities in placental anatomy between human and guinea pig. Furthermore, guinea pig CMV (GPCMV) has been demonstrated to cross the placenta and cause fetal infection and loss, similar to the effects of infection with HCMV. However, the kinetics of maternal and fetal infection in this model has not been carefully investigated. In this work, we have delineated the kinetics of maternal to fetal infection and found that congenital infection is rapid following maternal infection. Importantly, we demonstrate that a monoclonal antibody against a protein critical for viral entry protects pregnant guinea pigs against fetal infection. Thus, our studies may be informative for development of a therapeutic intervention to treat congenital HCMV infection in humans.

Immunoglobulin G (IgG) Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. A high resolution map of the binding site on human IgG1 for human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and FcRn receptors has been determined. A common set of IgG1 residues is involved in binding to all FcγR; FcγRII and FcγRIII also utilize residues outside this common set. In addition to residues which, when altered, abrogated binding to one or more of the receptors, several residues were found that improved binding only to specific receptors or simultaneously improved binding to one type of receptor and reduced binding to another type. Select IgG1 variants with improved binding to FcγRIIIA exhibited up to 100%enhancement in antibody-dependent cell cytotoxicity using human effector cells; these variants included changes at residues not found at the binding interface in the IgG/FcγRIIIA co-crystal structure (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000)Nature 406, 267–273). These engineered antibodies may have important implications for improving antibody therapeutic efficacy.

Vascular endothelial growth factor (VEGF) is a pleiotropic factor that exerts a multitude of biological effects through its interaction with two receptor tyrosine kinases,fms-like tyrosine kinase (Flt-1) or VEGF receptor 1 and kinase insert domain-containing receptor (KDR) or VEGF receptor 2. Whereas it is commonly accepted that KDR is responsible for the proliferative activities of VEGF, considerable controversy and uncertainty exist about the role of the individual receptors in eliciting many of the other effects. Based on a comprehensive mutational analysis of the receptor-binding site of VEGF, an Flt-1-selective variant was created containing four substitutions from the wild-type protein. This variant bound with wild-type affinity to Flt-1, was at least 470-fold reduced in binding to KDR, and had no activity in cell-based assays measuring autophosphorylation of KDR or proliferation of primary human vascular endothelial cells. Using a competitive phage display strategy, two KDR-selective variants were discovered with three and four changes from wild-type, respectively. Both variants had approximately wild-type affinity for KDR, were about 2000-fold reduced in binding to Flt-1, and showed activity comparable with the wild-type protein in KDR autophosphorylation and endothelial cell proliferation assays. These variants will serve as useful reagents in elucidating the roles of Flt-1 and KDR.

Vascular endothelial growth factor (VEGF) is a specific and potent angiogenic factor and, therefore, a prime therapeutic target for the development of antagonists for the treatment of cancer. As a first step toward this goal, phage display was used to generate peptides that bind to the receptor-binding domain (residues 8-109) of VEGF and compete with receptor [Fairbrother, W. J., Christinger, H. W., Cochran, A. G., Fuh, G., Keenan, C. J., Quan, C., Shriver, S. K., Tom, J. Y. K., Wells, J. A., and Cunningham, B. C. (1999) Biochemistry 38, 17754-17764]. The crystal structure of VEGF in complex with one of these peptides was solved and refined to a resolution of 1.9 A. The 20-mer peptide is unstructured in solution and adopts a largely extended conformation when bound to VEGF. Residues 3-8 form a beta-strand which pairs with strand beta6 of VEGF via six hydrogen bonds. The C-terminal four residues of the peptide point away from the growth factor, consistent with NMR data indicating that these residues are flexible in the complex in solution. In contrast, shortening the N-terminus of the peptide leads to decreased binding affinities. Truncation studies show that the peptide can be reduced to 14 residues with only moderate effect on binding affinity. However, because of the extended conformation and the scarcity of specific side-chain interactions with VEGF, the peptide is not a promising lead for small-molecule development. The interface between the peptide and VEGF contains a subset of the residues recognized by a neutralizing Fab fragment and overlaps partially with the binding site for the Flt-1 receptor. The location of the peptide-binding site and the hydrophilic character of the interactions with VEGF resemble more the binding mode of the Fab fragment than that of the receptor.

Although angiogenesis inhibitors have provided substantial clinical benefit as cancer therapeutics, their use is limited by resistance to their therapeutic effects. While ample evidence indicates that such resistance can be influenced by the tumor microenvironment, the underlying mechanisms remain incompletely understood. Here, we have uncovered a paracrine signaling network between the adaptive and innate immune systems that is associated with resistance in multiple tumor models: lymphoma, lung and colon. Tumor-infiltrating T helper type 17 (T(H)17) cells and interleukin-17 (IL-17) induced the expression of granulocyte colony-stimulating factor (G-CSF) through nuclear factor κB (NF-κB) and extracellular-related kinase (ERK) signaling, leading to immature myeloid-cell mobilization and recruitment into the tumor microenvironment. The occurrence of T(H)17 cells and Bv8-positive granulocytes was also observed in clinical tumor specimens. Tumors resistant to treatment with antibodies to VEGF were rendered sensitive in IL-17 receptor (IL-17R)-knockout hosts deficient in T(H)17 effector function. Furthermore, pharmacological blockade of T(H)17 cell function sensitized resistant tumors to therapy with antibodies to VEGF. These findings indicate that IL-17 promotes tumor resistance to VEGF inhibition, suggesting that immunomodulatory strategies could improve the efficacy of anti-angiogenic therapy.

Thrombopoietin (TPO) is the natural regulator of platelet production in the bone marrow of mammals. This cytokine also seems to play an important role in the development of the erythroid lineage when recovering from anemic conditions. Here we study the effects of various TPO molecules on the recovery of hematopoietic lineages in a mouse model of pancytopenia. Based on previous animal experimentation and clinical experience with other hematopoietic cytokines, we found that daily dosing with TPO augmented the recovery of both the megakaryocyte and erythroid lineages in a mouse model of pancytopenia. However, further experiments showed that no benefit was gained by using more than a single dose of recombinant murine (rm)TPO(335) given 24 h after the initiation of the myelosuppressive treatment. This response to a single dose of rmTPO(335) is dose-dependent. However, the response was attenuated when a truncated, short half-life TPO molecule (rmTPO[153]) was used. Increasing the half-life of the molecule with 10 kDa polyethylene glycol (PEG) does not improve the response. Only when larger PEG molecules (20 kDa or 40 kDa) are linked to the rmTPO(153) is the response to single doses restored to the level of the full-length molecule. These data suggest that, unlike our experience with other cytokines, the commitment of progenitors to a megakaryocytic cell line is accomplished by a single short exposure to TPO.

Recombinant human (rh) factor VIII is a glycoprotein consisting of multiple polypeptides with relative mobilities ( M r ) ranging from 80,000 to 210,000. It is produced in mammalian cells. Single-dose intravenous pharmacokinetic studies were conducted with rh factor VIII (Kogenate rh Antihemophilic Factor, Miles, Inc.) in male mice (21.0–25.8 g) and rats (252.0–254.2 g). Each species received 400 IU/kg, and blood was collected up to 12 hr (mice) or 32.5 hr (rats) post-dose. Immunoreactive factor VIII concentrations in plasma were quantified by a sensitive and specific ELISA. In both species, the disposition profiles were described by the sum of two exponentials. The pharmacokinetics of rh factor VIII in mouse were as follows: clearance, 27.7 ml/hr/kg; initial volume of distribution, 72 ml/kg; steady-state volume of distribution, 148 ml/kg; and terminal half-life, 4.1 hr. In rat, the mean estimates were as follows: clearance, 16.0 ml/hr/kg; initial volume of distribution, 41 ml/kg; steady-state volume of distribution, 125 ml/kg; and terminal half-life, 5.5 hr. These pharmacokinetic parameters for rh factor VIII in animals and human rh factor VIII pharmacokinetic parameters from the literature were evaluated to determine if the parameters can be represented by the allometric relationship, Y = aW b , where Y is the pharmacokinetic parameter, and W is body weight. The following allometric relations were obtained for rh factor VIII: clearance (ml/hr) = 10.4 W 0.69 , half-life (hr) = 7.5 W 0.18 , initial volume of distribution (ml) = 43.6 W 1.04 , and steady-state volume of distribution (ml) = 99.1 W 0.84 . The allometric exponents for each parameter conformed to theory and were within the range of values commonly observed for xenobiotics and therapeutic proteins. These studies suggest that the pharmacokinetics of rh factor VIII in laboratory animals are predictive of the disposition in humans despite the complex nature of its biological interactions and the chemical diversity of the purified material.

Antibodies as therapeutic agents have gained broad acceptance as shown by the number of antibodies in clinical use and many more in clinical development. This utility is an outcome of the high specificity and affinity of the antigen-binding site comprised of the heavy and light chain variable domains. In addition, the Fc portion of human or humanized IgG(1) antibodies promotes long half-life through interaction with the recycling FcRn receptor and effects killing functions through interaction with complement and Fcγ receptors. Engineering the Fc portion to increase half-life through stronger binding to FcRn, or to increase complement or cell-mediated killing may lead to improved therapeutic antibodies. These improvements may benefit the patients through convenience in dosing or increased efficacy. Here we describe protocols for generating Fc-engineered IgG(1) antibodies and assays to measure Fc receptor binding, antibody dependent cellular cytotoxicity activity, and complement dependent cytotoxicity activity to identify variants with improved FcRn binding or effector function.

Short interfering RNA (siRNA) has therapeutic potential. However, efficient delivery is a formidable task. To facilitate delivery of siRNA into cells, we covalently conjugated siRNA to antibodies that bind to cell surface proteins and internalize. Understanding how these antibody-siRNA conjugates function in vivo requires pharmacokinetic analysis. Thus, we developed a simple real-time antigen capture reverse transcription-polymerase chain reaction (RT-PCR) assay to detect intact antibody-siRNA conjugates. Biotinylated antigen bound to streptavidin-coated PCR tubes was used to capture antibody-siRNA conjugate. The captured antibody-siRNA conjugate was then reverse-transcribed in the same tube, avoiding a sample transfer step. This reproducible assay had a wide standard curve range of 0.029 to 480ng/ml and could detect as low as 0.58ng/ml antibody-siRNA conjugates in mouse serum. The presence of unconjugated antibody that could be generated from siRNA degradation in vivo did not affect the assay as long as the total antibody concentration in the antigen capture step did not exceed 480ng/ml. Using this assay, we observed a more rapid decrease in serum antibody-siRNA conjugate concentrations than the total antibody concentrations in mice dosed with antibody-siRNA conjugates, suggesting loss of siRNA from the antibody. This assay is useful for optimizing antibody-siRNA and likely aptamer-siRNA conjugates to improve pharmacokinetics and aid siRNA delivery.