Neutralization of the Staphylococcus aureus Panton-Valentine leukocidin by African and Caucasian sera.

BACKGROUND: The prevalence of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin (PVL) gene is higher in Africa (≈50%) compared to Europe (< 5%). The study aimed to measure anti-PVL-antibodies in Africans and Germans in a multi-center study and to test whether detected antibodies can neutralize the cytotoxic effect of PVL on polymorphonuclear leukocytes (PMNs). METHODS: Sera from asymptomatic Africans (n = 22, Nigeria, Gabon) and Caucasians (n = 22, Germany) were used to quantify antibody titers against PVL and α-hemolysin (in arbitrary units [AU]) by ELISA. PMNs from one African and German donor were exposed to 5 nM recombinant PVL to measure the neutralizing effect of serial dilutions of pooled sera from African and Caucasian participants, or donor sera at 0.625 and 2.5% (v/v). RESULTS: Anti-PVL-antibodies were significantly higher in Africans than in Germans (1.9 vs. 0.7 AU, p < 0.0001). The pooled sera from the study participants neutralized the cytotoxic effect of PVL on African and German PMNs in a dose dependent manner. Also, neutralization of PVL on PMNs from the African and German donors had a stronger effect with African sera (half-maximal inhibitory concentration (IC50) = 0.27 and 0.47%, respectively) compared to Caucasian sera (IC50 = 3.51 and 3.59% respectively). CONCLUSION: Africans have higher levels of neutralizing anti-PVL-antibodies. It remains unclear if or at what level these antibodies protect against PVL-related diseases.

Fit-for-purpose LC-MS/MS quantification of leukotoxin and leukotoxin diol in mouse plasma without sample pre-concentration.

LC/MS quantification of leukotoxin (LTX) and leukotoxin diol (LTXdiol) in plasma has been previously reported, however large sample volumes are required for achieving stated assay Lower Limit of Quantification (LLOQ). Reported here is a fit-for-purpose LC/MS method that reduces plasma volume from 700 to 25 µL and omits pre-concentration steps. These improvements make for a method with increased utility in mouse studies offering limited sample volumes. Additionally, omitting pre-concentration steps streamlines sample processing, which can now be completed in under 10 min. This method can be used to quickly answer if the ratio of LTX to LTXdiol changes with the dose of the therapeutic drug so this could be used as a potential biomarker for correlating PK/PD effects. No extensive assay characterization was performed before application to an exploratory in-life study. Basal levels of LTX and LTXdiol in plasma were quantified by LC-MRM across 10 individual mice, and the average signal-to-noise was 36 for LTX and 3039 for LTXdiol, with CVs of 29.4% and 15.2%, respectively. Addition of LTX and LTXdiol reference standard at 5, 25, and 75 ng/mL into pooled mouse plasma was quantifiable within 30% relative error using a surrogate matrix calibration curve ranging from 0.8 to 200 ng/mL. The average ratio of LTX to LTXdiol across the 10 mice was 0.32, consistent with previous reports. Finally, the method was applied to a mouse PK/PD study to monitor LTX/LTXdiol kinetics after a single oral dose of a soluble epoxide hydrolase inhibitor. The mean plasma ratio of LTX to LTXdiol increased up to 10-fold by 3 h post-dose followed by a decrease to near pre-dose levels by 24 h, consistent with transient inhibition of sEH-mediated conversion of LTX to LTXdiol. The method improvements described here will make subsequent quantification of LTX and LTXdiol in mouse studies significantly easier.

Osteomyelitis, Venous Thrombosis, and Septic Emboli in a Pediatric Patient: A Case Report.

New virulence factors, such as the Panton-Valentine leukocidin (PVL), are appearing during Staphylococcus aureus infections occurring in the pediatric population. Such factors increase the aggressiveness and risk of dissemination of the bacteria, causing infections to be life-threatening. An early diagnosis is thus especially important. We present a case of osteomyelitis, venous thrombosis, and septic emboli occurring in a pediatric patient that should trigger suspicion of a PVL-positive strain. A multidisciplinary approach is necessary to enable rapid diagnosis and early treatment, which is essential for successful management of these infections. Management is based on broad-spectrum antibiotics, in combination with aggressive surgical treatment and antithrombotic therapy. In patients infected with S. aureus whose condition worsens quickly, PVL gene sequencing should be considered.

Staphylococcus aureus Pneumonia in the Community.

Staphylococcus aureus is an emergent etiology of community-acquired pneumonia (CAP) over the past 2 decades, with severe community-acquired pneumonia (SCAP) caused by methicillin-resistant S. aureus (MRSA) leading to critical illness and death. S. aureus colonization is associated with a high incidence of pneumonia. Panton-Valentine leukocidin (PVL) is one of the most important virulence factors of S. aureus associated with serious complications. In recent years, community-associated MRSA (CA-MRSA) clones that caused infections in young adults and healthy individuals with no exposure to health care settings and no classical risk factors have emerged. Clinical features at admission including concurrent influenza infection, hemoptysis, multilobar infiltrates, and neutropenia should suggest S. aureus CAP. Sputum Gram stains, cultures (or tracheobronchial aspirates or bronchoalveolar lavage in mechanically ventilated patients), polymerase chain reaction (nasopharyngeal or oropharyngeal or lower respiratory tract specimens), and two sets of blood cultures should be obtained from patients presenting with severe S. aureus CAP. For CAP due to methicillin-susceptible S. aureus, first-line therapy is usually cefazolin, oxacillin, or ceftaroline. For CA-MRSA pneumonia, linezolid is recommended. If vancomycin or teicoplanin are used, combination with clindamycin or rifampicin should be considered in cases of PVL-positive MRSA CAP.

Clinical Manifestations in Children with Staphylococcal Bacteremia Positive for Panton-Valentine Leucocidin: A Nationwide Survey in a Low Methicillin-Resistance Setting.

A total of 714 pediatric cases of Staphylococcus aureus bacteremia were identified from 2008 to 2015 in Denmark; 98% were methicillin-susceptible S. aureus (MSSA). Fifteen isolates (2,1%) were Panton-Valentine leucocidin positive (0.17/100,000 children/year) and 87% MSSA. Eight cases (53%) were severe, including all pneumonia cases. Panton-Valentine leucocidin positive Staphylococcus aureus bacteremia is rare in our setting with high MSSA-prevalence. Half of the cases were uncomplicated.

Prevalence, clinical expression, invasiveness and outcome of Staphylococcus aureus containing Panton-Valentine leukocidin in children treated in a university hospital of Lithuania.

Background: There is a high prevalence of Staphylococcus aureus virulence factor Panton-Valentine leukocidin (PVL) in North-East parts of Europe. The aim was to evaluate data regarding the PVL occurrences in Lithuania, determine the relationship with Methicillin resistant Staphylococcus aureus (MRSA), association with demographic and clinical conditions, invasiveness and severity of the disease in children treated in hospital Kauno klinikos (KK).Methods: We performed a prospective case-cohort single-center study on paediatric patients hospitalized from 2012 to 2015 to KK. We compared characteristics in PVL positive [SA-PVL(+)] and PVL negative [SA-PVL(-)] groups among non-invasive and invasive infections. Logistic regression was performed to detect PVL predicting factors and Cox regression was presented to define factors associated with admission to intensive care unit (ICU).Results: PVL was detected in 51.5%, MRSA in 7.0% and MRSA-PVL(+) in 4.8% of cases. In general, PVL was associated with older age comparing with SA-PVL(-) (median 8.5 vs. 4.0 years, p < .001). Skin and soft tissue infections were presented in 87.9% of all SA-PVL(+) cases. Invasive infections (44.7% vs. 12.1%, p < .001) and co-morbidities (20.5% vs. 2.9%, p < .001) were associated with SA-PVL(-) infections compared to SA-PVL(+), but ICU admission number was higher in invasive SA-PVL(+) cases comparing to invasive SA-PVL(-) cases (41.2% vs. 10.2%, p = .007).Conclusions: There was a high prevalence of pvl gene in patients treated in KK. SA-PVL(+) infections were associated with SSTI and were not common in invasive infections, but the invasive infections caused by SA-PVL(+) were related to severe disease progression and admission to ICU.

Clindamycin clearance during Cytosorb® hemoadsorption: A case report and pharmacokinetic study.

Panton-Valentine leucocidin producing methicillin-resistant Staphylococcus aureus infections are rare but associated with very high mortality rates. We report the case of a 14-year-old patient with Panton-Valentine leucocidin producing methicillin-resistant Staphylococcus aureus infection and Influenza B pneumonia requiring veno-arterial extra-corporeal membrane oxygenator for refractory shock. In the absence of response to conventional therapy, we have inserted a Cytosorb® cartridge within the extra-corporeal membrane oxygenator circuit. A spectacular decrease in vasopressor requirements followed. Since clindamycin, a key component of Panton-Valentine leucocidin producing methicillin-resistant Staphylococcus aureus treatment, might be removed by Cytosorb® hemoadsorption, we have performed serial plasma concentrations measurements of the drug. Based on these measurements, we were able to develop a pharmacokinetic model incorporating variable plasma clearance. Patient's exposure was estimated before, during and after Cytosorb® hemoadsorption. According to this model, Cytosorb® hemoadsorption did not seem to result in significant clindamycin removal. Cytosorb® hemoadsorption during Panton-Valentine leucocidin producing methicillin-resistant Staphylococcus aureus infection appears safe and feasible and no adaptation of clindamycin dosage seems necessary.

Effective concentration of intravenous immunoglobulin for neutralizing Panton-Valentine leukocidin in human blood.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infects healthy individuals, although the precise cause remains unclear. CA-MRSA produces Panton-Valentine leukocidin (PVL), which often causes severe invasive infection; however, antitoxin drugs against PVL are limited. Intravenous immunoglobulin (IVIg) possesses antitoxin activity, but unfortunately, the optimal dose is unknown. Here, we measured the PVL neutralizing antibody titer in the plasma of Japanese individuals and sera of American donors. Next, we compared the cytotoxic effects of PVL on neutrophils in phosphate buffered saline (PBS) or whole blood to determine the effect of the neutralizing antibody. Finally, we evaluated the effective concentration of IVIg required to neutralize PVL in PBS and whole blood. We observed that the titer of PVL neutralizing antibody in healthy individuals polarized as high and low/none group. Additionally, the PVL neutralizing antibody titer considerably affected the concentration at which IVIg elicited its effect. This suggests that PVL-producing CA-MRSA might be involved in determining the severity of infection in healthy individuals without neutralizing antibody against PVL. The neutralizing effect of IVIg was observed in both PBS and whole blood. However, the optimal concentration of IVIg required for neutralizing PVL varied between PBS and whole blood. In addition, since the PVL-neutralizing activity of IVIg also largely depends on blood composition, such as neutralizing antibody concentration, the optimal dosage of IVIg as an antitoxin drug should be decided in a timely manner after considering the patient's medical background.

Protection of the Furin Cleavage Site in Low-Toxicity Immunotoxins Based on Pseudomonas Exotoxin A.

Recombinant immunotoxins (RITs) are fusions of an Fv-based targeting moiety and a toxin. Pseudomonas exotoxin A (PE) has been used to make several immunotoxins that have been evaluated in clinical trials. Immunogenicity of the bacterial toxin and off-target toxicity have limited the efficacy of these immunotoxins. To address these issues, we have previously made RITs in which the Fv is connected to domain III (PE24) by a furin cleavage site (FCS), thereby removing unneeded sequences of domain II. However, the PE24 containing RITs do not contain the naturally occurring disulfide bond around the furin cleavage sequence, because it was removed when domain II was deleted. This could potentially allow PE24 containing immunotoxins to be cleaved and inactivated before internalization by cell surface furin or other proteases in the blood stream or tumor microenvironment. Here, we describe five new RITs in which a disulfide bond is engineered to protect the FCS. The most active of these, SS1-Fab-DS3-PE24, shows a longer serum half-life than an RIT without the disulfide bond and has the same anti-tumor activity, despite being less cytotoxic in vitro. These results have significance for the production of de-immunized, low toxicity, PE24-based immunotoxins with a longer serum half-life.

Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum.

Exotoxin A is one of the virulence factors of Pseudomonas aeruginosa, a bacterium that can cause infections resulting in adverse health outcomes and increased burden to health care systems. Current methods of diagnosing P. aeruginosa infections are time consuming and can require significant preparation of patient samples. This study utilized a novel variation of the Systematic Evolution of Ligand by Exponential Enrichment, Decoy-SELEX, to identify an Exotoxin A specific single-stranded DNA (ssDNA) molecular recognition element (MRE). Its emphasis is on increasing stringency in directing binding toward free target of interest and at the same time decreasing binding toward negative targets. A ssDNA MRE with specificity and affinity was identified after fourteen rounds of Decoy-SELEX. Utilizing surface plasmon resonance measurements, the determined equilibrium dissociation constant (Kd ) of the MRE is between 4.2 µM and 4.5 µM, and is highly selective for Exotoxin A over negative targets. A ssDNA MRE modified sandwich enzyme-linked immunosorbent assay (ELISA) has been developed and achieved sensitive detection of Exotoxin A at nanomolar concentrations in human serum. This study has demonstrated the proof-of-principle of using a ssDNA MRE as a clinical diagnostic tool.

The role of serum Pseudomonas aeruginosa antibodies in the diagnosis and follow-up of cystic fibrosis.

In cystic fibrosis (CF), if Pseudomonas aeruginosa (Pa) infection is not diagnosed and treated early, chronic colonization occurs, which causes rapid decline in pulmonary functions. The aim of this study was to evaluate Pa antibodies, compare them with Pa cultures and determine their role in early diagnosis and follow-up. Ninety CF patients were included; they were divided into chronic, intermittent, negative, and mucoid groups. They were evaluated every 3-6 months. In each visit, pulmonary function tests and sputum cultures were obtained, and Pa antibodies exotoxin A (ExoA), elastase (ELA) and alkaline protease (AP) were determined in the serum by enzyme-linked immunosorbent assay (ELISA). The most specific test that discriminated chronic colonized patients from noncolonized patients was Pa culture, and the presence of at least one antibody had the highest sensitivity. AP had the highest specificity, and ELA had the highest sensitivity. All antibodies were highest in the mucoid group. ELA was highest in chronic and lowest in the negative group. The presence of antibodies was much higher than positive Pa cultures in patients younger than five years of age. A negative correlation between forced expiratory volume in 1 second (FEV1) and AP was determined only in the mucoid group. In the two-year follow-up, antibody presence did not show a regular pattern. In CF, Pa antibodies can be early markers for diagnosis, especially in young children who cannot expectorate, but they should only be used together with sputum cultures for long-term follow-up and treatment.

Pharmacokinetic and pharmacodynamic comparability study of moxetumomab pasudotox, an immunotoxin targeting CD22, in cynomolgus monkeys.

Moxetumomab pasudotox is an immunotoxin currently being investigated in patients for the treatment of CD22-expressing B-cell malignancies. A single-cycle pharmacokinetic (PK)-pharmacodynamic (PD) study was conducted in cynomolgus monkeys for PK comparability assessment and population PK-PD modeling after major manufacturing process and site changes. Primates were randomized by body weight and baseline CD22 lymphocyte counts to receive intravenous administrations of 1 mg/kg moxetumomab pasudotox (n = 12/group) on Days 1, 3, and 5. PK and B-lymphocyte count data were modeled using a population approach. The 90% confidence intervals of the geometric mean ratios of PK exposure were within the 80%-125% range. The B lymphocytes were depleted to a similar extent, and the immunogenicity incidences were similar across the two groups. The B-cell depletion was described by a novel lifespan model in which moxetumomab pasudotox induced random destruction of B cells in each aging compartment. The endogenous de novo influx from bone marrow was subject to a negative feedback mechanism. The estimated B cell apparent lifespan was 51 days. Covariate analysis confirmed that the manufacturing change had no impact on PK or PD of moxetumomab pasudotox. Results from this study supported continued clinical investigation of moxetumomab pasudotox using the new material.

[Is monotherapy with ß-lactam antibiotics still up to date? New aspects for treatment of severe infections]. / Sind Monotherapien mit ß-Laktamantibiotika noch zeitgemäss?

Mortality of sepsis is still high. Crucial for therapeutic response are the early start of treatment as well as the choice of antibiotics or antibiotic combinations. ß-lactam antibiotics with bactericidal mode of action are often recommended in guidelines. But this antibiotic class can trigger the immune system to a maximum by releasing cell wall components or exotoxins. This may lead to a worsening of the patient's clinical situation. In contrast, antibiotics with bacteriostatic action often inhibit bacterial protein synthesis with decrease of production of virulence factors and minimize release of cell wall components. The purpose of this review is to summarise the significance of some bacteriostatic antibiotics and to discuss whether a combination of bactericidal and bacteriostatic agents may improve the course of the illness.

Cutaneous pustular manifestations associated with disseminated septic embolism due to a Panton-Valentine leukocidin-producing strain of community-acquired methicillin-resistant Staphylococcus aureus.

We report a 12-year-old child presented with cutaneous pustular lesions. The lesions are associated with disseminated septic embolism due to Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The mortality of CA-MRSA sepsis associated with cutaneous findings is high. In areas where CA-MRSA is endemic, empiric treatment of suspected cutaneous manifestations should include antibiotics predictably active against this pathogen.

[Panton-Valentine leukocidin positive Staphylococcus aureus isolated from blood in Korea].

BACKGROUND: Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by some Staphylococcus aureus strains and associated with skin and soft tissue infections; these strains are epidemiologically associated with current outbreaks of community-acquired methicillin-resistant S. aureus (MRSA) and with necrotizing pneumonia in healthy adults in USA and Europe. This study was performed to investigate the presence of PVL-positive S. aureus and the significant infections known to be caused by this organism. METHODS: A total of 573 strains of S. aureus blood isolates at university-affiliated hospital during 2002 to 2005 were selected. The presence of PVL was investigated using PCR. Additional 12 staphylococcal toxin genes were also examined in PVL-positive S. aureus strains, and MRSA isolates were typed for the staphylococcal cassette chromosome mec (SCCmec). RESULTS: PVL genes were detected in 5 (0.9%) of 573 S. aureus strains, including 1 MRSA and 4 MSSA. The PVL-positive MRSA isolate was SCCmec type IV, and no other staphylococcal toxins were detected. The median age of the patients infected with PVL-positive S. aureus was 36 yr. Three cases of bacteremia were preceded by skin and soft-tissue infections. CONCLUSIONS: Bacteremia caused by PVL-positive S. aureus strain were detected in 5 patients in Korea, and some of the patients were associated with severe skin and soft-tissue infections. In addition, the PVL-positive MRSA strain of SCCmec type IV, a characteristic of community-acquired MRSA isolates in USA and Europe, also exists in Korea, and can cause the severe infections known to be associated with this organism.

A case of naturally acquired inhalation anthrax: clinical care and analyses of anti-protective antigen immunoglobulin G and lethal factor.

This report describes the first case of naturally acquired inhalation anthrax in the United States since 1976. The patient's clinical course included adjunctive treatment with human anthrax immunoglobulin. Clinical correlation of serologic assays for the lethal factor component of lethal toxin and anti-protective antigen immunoglobulin G are also presented.

Panton-Valentine Leukocidin Positive Staphylococcus aureus Isolated from Blood in Korea / 대한진단검사의학회지

BACKGROUND: Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by some Staphylococcus aureus strains and associated with skin and soft tissue infections; these strains are epidemiologically associated with current outbreaks of community-acquired methicillin-resistant S. aureus (MRSA) and with necrotizing pneumonia in healthy adults in USA and Europe. This study was performed to investigate the presence of PVL-positive S. aureus and the significant infections known to be caused by this organism. METHODS: A total of 573 strains of S. aureus blood isolates at university-affiliated hospital during 2002 to 2005 were selected. The presence of PVL was investigated using PCR. Additional 12 staphylococcal toxin genes were also examined in PVL-positive S. aureus strains, and MRSA isolates were typed for the staphylococcal cassette chromosome mec (SCCmec). RESULTS: PVL genes were detected in 5 (0.9%) of 573 S. aureus strains, including 1 MRSA and 4 MSSA. The PVL-positive MRSA isolate was SCCmec type IV, and no other staphylococcal toxins were detected. The median age of the patients infected with PVL-positive S. aureus was 36 yr. Three cases of bacteremia were preceded by skin and soft-tissue infections. CONCLUSIONS: Bacteremia caused by PVL-positive S. aureus strain were detected in 5 patients in Korea, and some of the patients were associated with severe skin and soft-tissue infections. In addition, the PVL-positive MRSA strain of SCCmec type IV, a characteristic of community-acquired MRSA isolates in USA and Europe, also exists in Korea, and can cause the severe infections known to be associated with this organism.

Panton-Valentine Leukocidin Positive Staphylococcus aureus Isolated from Blood in Korea / 대한진단검사의학회지

BACKGROUND: Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by some Staphylococcus aureus strains and associated with skin and soft tissue infections; these strains are epidemiologically associated with current outbreaks of community-acquired methicillin-resistant S. aureus (MRSA) and with necrotizing pneumonia in healthy adults in USA and Europe. This study was performed to investigate the presence of PVL-positive S. aureus and the significant infections known to be caused by this organism. METHODS: A total of 573 strains of S. aureus blood isolates at university-affiliated hospital during 2002 to 2005 were selected. The presence of PVL was investigated using PCR. Additional 12 staphylococcal toxin genes were also examined in PVL-positive S. aureus strains, and MRSA isolates were typed for the staphylococcal cassette chromosome mec (SCCmec). RESULTS: PVL genes were detected in 5 (0.9%) of 573 S. aureus strains, including 1 MRSA and 4 MSSA. The PVL-positive MRSA isolate was SCCmec type IV, and no other staphylococcal toxins were detected. The median age of the patients infected with PVL-positive S. aureus was 36 yr. Three cases of bacteremia were preceded by skin and soft-tissue infections. CONCLUSIONS: Bacteremia caused by PVL-positive S. aureus strain were detected in 5 patients in Korea, and some of the patients were associated with severe skin and soft-tissue infections. In addition, the PVL-positive MRSA strain of SCCmec type IV, a characteristic of community-acquired MRSA isolates in USA and Europe, also exists in Korea, and can cause the severe infections known to be associated with this organism.

Phase I clinical study of the recombinant antibody toxin scFv(FRP5)-ETA specific for the ErbB2/HER2 receptor in patients with advanced solid malignomas.

INTRODUCTION: ScFv(FRP5)-ETA is a recombinant antibody toxin with binding specificity for ErbB2 (HER2). It consists of an N-terminal single-chain antibody fragment (scFv), genetically linked to truncated Pseudomonas exotoxin A (ETA). Potent antitumoral activity of scFv(FRP5)-ETA against ErbB2-overexpressing tumor cells was previously demonstrated in vitro and in animal models. Here we report the first systemic application of scFv(FRP5)-ETA in human cancer patients. METHODS: We have performed a phase I dose-finding study, with the objective to assess the maximum tolerated dose and the dose-limiting toxicity of intravenously injected scFv(FRP5)-ETA. Eighteen patients suffering from ErbB2-expressing metastatic breast cancers, prostate cancers, head and neck cancer, non small cell lung cancer, or transitional cell carcinoma were treated. Dose levels of 2, 4, 10, 12.5, and 20 microg/kg scFv(FRP5)-ETA were administered as five daily infusions each for two consecutive weeks. RESULTS: No hematologic, renal, and/or cardiovascular toxicities were noted in any of the patients treated. However, transient elevation of liver enzymes was observed, and considered dose limiting, in one of six patients at the maximum tolerated dose of 12.5 microg/kg, and in two of three patients at 20 microg/kg. Fifteen minutes after injection, peak concentrations of more than 100 ng/ml scFv(FRP5)-ETA were obtained at a dose of 10 microg/kg, indicating that predicted therapeutic levels of the recombinant protein can be applied without inducing toxic side effects. Induction of antibodies against scFv(FRP5)-ETA was observed 8 days after initiation of therapy in 13 patients investigated, but only in five of these patients could neutralizing activity be detected. Two patients showed stable disease and in three patients clinical signs of activity in terms of signs and symptoms were observed (all treated at doses > or = 10 microg/kg). Disease progression occurred in 11 of the patients. CONCLUSION: Our results demonstrate that systemic therapy with scFv(FRP5)-ETA can be safely administered up to a maximum tolerated dose of 12.5 microg/kg in patients with ErbB2-expressing tumors, justifying further clinical development.