Kyu Hong

VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism

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.

Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies.

Many research and clinical applications require large quantities of full-length antibodies with long circulating half-lives, and production of these complex multi-subunit proteins has in the past been restricted to eukaryotic hosts. In this report, we demonstrate that efficient secretion of heavy and light chains in a favorable ratio leads to the high-level expression and assembly of full-length IgGs in the Escherichia coli periplasm. The technology described offers a rapid, generally applicable and potentially inexpensive method for the production of full-length therapeutic antibodies, as verified by the expression of several humanized IgGs. One E. coli-derived antibody in particular, anti-tissue factor IgG1, has been thoroughly evaluated and has all of the expected properties of an aglycosylated antibody, including tight binding to antigen and the neonatal receptor. As predicted, the protein lacks binding to C1q and the FcgammaRI receptor, making it an ideal candidate for research purposes and therapeutic indications where effector functions are either not required or are actually detrimental. In addition, a limited chimpanzee study suggests that the E. coli-derived IgG1 retains the long circulating half-life of mammalian cell-derived antibodies.

Association between HER-2/neu and Vascular Endothelial Growth Factor Expression Predicts Clinical Outcome in Primary Breast Cancer Patients

Purpose: Activation or overexpression of HER-2/neu is associated with up-regulation of vascular endothelial growth factor (VEGF) in human breast cancer cells in vitro. Preclinical experiments indicate that increased expression of VEGF may in part mediate the biologically aggressive phenotype of HER-2/neu-overexpressing human breast cancer. It was the purpose of this study to: (a) evaluate the association between HER-2/neu and VEGF expression in a large clinical cohort of primary breast cancer patients; (b) compare the prognostic significance of VEGF isoforms; and (c) analyze the combined effects of HER-2/neu and VEGF on clinical outcome. Experimental Design: HER-2/neu and VEGF were measured by ELISA in primary breast tumor tissue lysates from 611 unselected patients with a median clinical follow-up of 50 months. At least six VEGF isoforms consisting of 121, 145, 165, 183, 189, or 206 amino acids are generated as a result of alternative splicing. The VEGF121–206 ELISA uses antibodies that bind to VEGF121 and, therefore, detects all of the VEGF isoforms with 121 and more amino acids. The VEGF165–206 ELISA uses antibodies that bind to VEGF165 and, therefore, detects all of the VEGF isoforms with 165 and more amino acids. VEGF121–206 and VEGF165–206 were analyzed both as continuous and categorical variables, using detectable expression as a cutoff for positivity. Cell lines with defined HER-2/neu expression levels were used to establish a cutoff point for HER-2/neu overexpression in breast tumor samples. Results: Our findings indicate a significant positive association between HER-2/neu and VEGF expression. VEGF121–206 and VEGF165–206 expression was detectable in 88 (77.2%) and 100 (87.7%), respectively, of the 114 patients with HER-2/neu-overexpressing tumors, in contrast to 271 (54.5%) and 353 (71.0%), respectively, of the 497 patients with nonoverexpressing tumors (χ2 test: P < 0.001 for both VEGF121–206 and VEGF165–206). VEGF121–206 and VEGF165–206 demonstrate a comparable prognostic significance for survival in unselected primary breast cancer patients (univariate analysis: VEGF121–206, P = 0.0068; VEGF165–206, P = 0.0046; multivariate analysis: VEGF121–206, P = 0.1475; VEGF165–206, P = 0.1483). When the analyses were performed separately for node-negative and node-positive patients, VEGF121–206 and VEGF165–206 were of prognostic significance for survival only in node-positive patients (univariate analysis: VEGF121–206, P = 0.0003; VEGF165–206, P = 0.0038; multivariate analysis: VEGF121–206, P = 0.0103; VEGF165–206, P = 0.0150). A biological concentration-effect relationship between VEGF expression and survival (VEGF121–206, P = 0.0280; VEGF165–206, P = 0.0097) suggests that VEGF levels, as determined by ELISA, could be of importance as a predictive marker for therapeutic strategies that target VEGF. Combining HER-2/neu and VEGF121–206/VEGF165–206 results in additional prognostic information for survival (VEGF121–206, P = 0.0133; VEGF165–206, P = 0.0092). Conclusion: The positive association between HER-2/neu and VEGF expression implicates VEGF in the aggressive phenotype exhibited by HER-2/neu overexpression, and supports the use of combination therapies directed against both HER-2/neu and VEGF for treatment of breast cancers that overexpress HER-2/neu.

Overcoming soluble target interference in an anti-therapeutic antibody screening assay for an antibody-drug conjugate therapeutic

BACKGROUND The standard safety evaluation of biotherapeutics includes assessment of immunogenicity. Anti-therapeutic antibodies (ATA) can be detected in serum using immunoassays with a bridging format. However, these assays can be subject to interference. RESULTS In the bridging ATA assay for 3A5 TDC, an antibody-drug conjugate that binds to the multimeric extracellular domain of MUC16 (CA125), soluble CA125 in the serum caused false-positive results by binding to the ATA assay reagents. This interaction was blocked by wheat germ agglutinin lectin as it binds to the glycans in CA125; thus, the specificity of the assay improved. CONCLUSION The assay development and validation results showed that the addition of wheat germ agglutinin eliminates the interference from circulating CA125 without impacting the ability to detect ATA.

Identification of IgG1 variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn: Comparison of soluble receptor-based and cell-based binding assays

Clinical response to the anti-CD20 antibody rituximab has been demonstrated to correlate with the polymorphism in the FcγRIIIa receptor where patients homozygous for the higher affinity V158 allotype showed a better response rate. This finding suggests that engineering of anti-CD20 for increased FcγRIIIa affinity could result in improved clinical outcome. To identify variants with increased affinity to FcγRIIIa, we developed quantitative assays using soluble receptors as well as engineered cell lines expressing FcγRI or FcγRIIIa on the cell surface. We assayed a set of anti-CD20 IgG(1) variants that had identical Fab regions, but alterations in the Fc regions, in both the soluble receptor-based and cell-based FcγRIIIa binding assays. We obtained similar relative binding affinity increases and assay precisions. The increase in affinity for FcγRIIIa correlated with the increase in activity in the antibody-dependent cellular cytotoxicity assay. These variants had unaltered FcγRI binding. In addition to Fcγ receptors, IgG also binds to FcRn, the receptor responsible for the long circulating half-life of IgG. The mutations in the anti-CD20 variants were previously found not to affect FcRn binding in the soluble receptor-based assays; consequently, we used anti-Her2 variants with different binding affinities to FcRn to study FcRn binding assays. We generated a cell line expressing FcRn on the cell surface to measure IgG binding and obtained similar ranking of these anti-Her2 variants in the cell-based and the soluble receptor-based FcRn binding assays. In conclusion, both the soluble receptor-based and cell-based binding assays can be used to identify IgG(1) variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn.

Pharmacokinetics and pharmacodynamics of DSTA4637A: A novel THIOMAB™ antibody antibiotic conjugate against Staphylococcus aureus in mice

ABSTRACT DSTA4637A, a novel THIOMAB™ antibody antibiotic conjugate (TAC) against Staphylococcus aureus (S. aureus), is currently being investigated as a potential therapy against S. aureus infections. Structurally, TAC is composed of an anti-S. aureus antibody linked to a potent antibiotic, dmDNA31. The goal of the current study was to characterize the pharmacokinetics (PK) of TAC in mice, assess the effect of S. aureus infection on its PK, and evaluate its pharmacodynamics (PD) by measuring the bacterial load in various organs at different timepoints following TAC treatment. Plasma concentrations of 3 analytes, total antibody (TAb), antibody-conjugated dmDNA31 (ac-dmDNA31), and unconjugated dmDNA31, were measured in these studies. In non-infected mice (target antigen absent), following intravenous (IV) administration of a single dose of TAC, systemic concentration-time profiles of both TAb and ac-dmDNA31 were bi-exponential and characterized by a short distribution phase and a long elimination phase as expected for a monoclonal antibody-based therapeutic. Systemic exposures of both TAb and ac-dmDNA31 were dose proportional over the dose range tested (5 to 50 mg/kg). In a mouse model of systemic S. aureus infection (target antigen present), a single IV dose of TAC demonstrated PK behavior similar to that in the non-infected mice, and substantially reduced bacterial load in the heart, kidney, and bones on 7 and 14 d post dosing. These findings have increased our understanding of the PK and PK/PD of this novel molecule, and have shown that at efficacious dose levels the presence of S. aureus infection had minimal effect on TAC PK.

A New ELISA for Use in a 3-ELISA System to Assess Concentrations of VEGF Splice Variants and VEGF110 in Ovarian Cancer Tumors

BACKGROUND Vascular endothelial growth factor (VEGF), which affects tumor angiogenesis, is expressed as different splice variants, including the major isoforms VEGF(165) and VEGF(121), and can be cleaved by plasmin to generate VEGF(110). The amount of VEGF(121) and VEGF(110) in biological samples has not been well studied. METHODS We developed an ELISA that detects VEGF(165) and VEGF(121) equally, but does not detect VEGF(110). We used this ELISA together with 2 other ELISAs, one detecting VEGF(165) and the other detecting VEGF(165), VEGF(121), and VEGF(110) equally, to assess the concentrations of VEGF(121) and VEGF(110) in ovarian cancer tumors. RESULTS The median concentrations in ovarian cancer tumor lysates were 0.61 (range <0.055-74) fmol/mg protein for VEGF(165), 1.4 (range <0.20-500) fmol/mg protein for VEGF(165) plus VEGF(121), and 2.3 (range <0.079-520) fmol/mg protein for total VEGF including VEGF(110) (n = 248). VEGF concentrations measured by the 3 ELISAs were highly correlated (r = 0.91-0.94). Median estimated VEGF(121) and VEGF(110) concentrations were 0.77 and 0.58 fmol/mg protein, respectively. In lysates with measurable VEGF(165) and total VEGF concentrations, mean VEGF(165) was approximately 31% (SD 23%) of the total VEGF (n = 217). In contrast, VEGF(165) constituted approximately half of the total circulating VEGF. CONCLUSION VEGF(165), VEGF(121), and VEGF(110) may be present at significant amounts in ovarian cancer tumors.

Commercial biomarker assays: friend and foe

AIM Commercial kits can provide a convenient solution for measuring circulating biomarkers to support drug development. However, their suitability should be assessed in the disease matrix of interest. METHODOLOGY Twelve biomarkers were evaluated in samples from patients with rheumatoid arthritis. We used immunoassay kits from five vendors on three multiplexed platforms. Kit suitability was evaluated on the basis of detectability and prespecified performance acceptance criteria. RESULTS Assays had varying levels of sensitivity and susceptibility to interference by matrix components. Only a few assays in the multiplexed kits were found to be suitable. In general, kits for analytes that passed our assay criteria showed good correlation between vendors. CONCLUSION The data from this study demonstrate that the majority of assays on multiplexed kits evaluated either lacked sensitivity and/or had poor performance, which diminishes the utility of the multiplexing approach.

Minimal physiologically-based pharmacokinetic modeling of DSTA4637A, A novel THIOMAB™ antibody antibiotic conjugate against Staphylococcus aureus, in a mouse model

ABSTRACT DSTA4637A, a THIOMAB™ antibody-antibiotic conjugate targeting Staphylococcus aureus, has shown promising bactericidal activity in a mouse model. DSTA4637A consists of a monoclonal anti-S. aureus antibody with an average of two rifalogue antibiotic molecules, dmDNA31, linked to its light chains. The goal of this study was to develop a minimal physiologically-based pharmacokinetic (mPBPK) model to characterize the pharmacokinetic (PK) properties of three analytes of DSTA4637A (i.e., total antibody, antibody-conjugated dmDNA31, and unconjugated dmDNA31) in mice, and to predict pharmacokinetics of DSTA4637A analytes in humans, as well as to provide an initial assessment for potential PK drug-drug interactions (DDI) in clinical trials via cross-species scaling of the mPBPK model. In the proposed model, selected organs, including heart, liver, and kidney, were connected anatomically with plasma and lymph flows. Mouse plasma and tissue concentrations of the three analytes of DSTA4637A were fitted simultaneously to estimate the PK parameters. Cross-species scaling of the model was performed by integrating allometric scaling and human physiological parameters. The final mPBPK model was able to successfully capture PK profiles of three DSTA4637A analytes in mouse plasma and in investigated organs. The model predicted a steady-state peak unbound dmDNA31 concentration lower than 5% of the IC50 of dmDNA31 towards cytochrome P450 following 100 mg/kg weekly intravenous dose, which suggests a low risk of PK DDI in humans for DSTA4637A with co-administered cytochrome P450 substrates. The proposed mPBPK modeling and cross-species scaling approaches provide valuable tools that facilitate the understanding and translation of DSTA4637A disposition from preclinical species to humans.

A Prognostic Model of Persistent Bacteremia and Mortality in Complicated Staphylococcus aureus Bloodstream Infection

BACKGROUND Staphylococcus aureus is a leading cause of bacteremia, yet there remains a significant knowledge gap in the identification of relevant biomarkers that predict clinical outcomes. Heterogeneity in the host response to invasive S. aureus infection suggests that specific biomarker signatures could be utilized to differentiate patients prone to severe disease, thereby facilitating earlier implementation of more aggressive therapies. METHODS To further elucidate the inflammatory correlates of poor clinical outcomes in patients with S. aureus bacteremia, we evaluated the association between a panel of blood proteins at initial presentation of bacteremia and disease severity outcomes using 2 cohorts of patients with S. aureus bacteremia (n = 32 and n = 124). RESULTS We identified 13 candidate proteins that were correlated with mortality and persistent bacteremia. Prognostic modeling identified interleukin (IL)-8 and CCL2 as the strongest individual predictors of mortality, with the combination of these biomarkers classifying fatal outcome with 89% sensitivity and 77% specificity (P < .0001). Baseline IL-17A levels were elevated in patients with persistent bacteremia (P < .0001), endovascular (P = .026) and metastatic tissue infections (P = .012). CONCLUSIONS These results demonstrate the potential utility of selected biomarkers to distinguish patients with the highest risk for treatment failure and bacteremia-related complications, providing a valuable tool for clinicians in the management of S. aureus bacteremia. Additionally, these biomarkers could identify patients with the greatest potential to benefit from novel therapies in clinical trials.