Alexander Berthold Hendrik Bakker

Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets.

Summary SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal antibody in ferrets, which can be readily infected with the virus. Prophylactic administration of the monoclonal antibody at 10 mg/kg reduced replication of SARS coronavirus in the lungs of infected ferrets by 3·3 logs (95% Cl 2·6–4·0 logs; p<0·001), completely prevented the development of SARS coronavirus-induced macroscopic lung pathology (p=0·013), and abolished shedding of virus in pharyngeal secretions. The data generated in this animal model show that administration of a human monoclonal antibody might offer a feasible and effective prophylaxis for the control of human SARS coronavirus infection.

C-Type Lectin-Like Molecule-1 A Novel Myeloid Cell Surface Marker Associated with Acute Myeloid Leukemia

Acute myeloid leukemia (AML) has a poor prognosis due to treatment-resistant relapses. A humanized anti-CD33 antibody (Mylotarg) showed a limited response rate in relapsed AML. To discover novel AML antibody targets, we selected a panel of single chain Fv fragments using phage display technology combined with flow cytometry on AML tumor samples. One selected single chain Fv fragment broadly reacted with AML samples and with myeloid cell lineages within peripheral blood. Expression cloning identified the antigen recognized as C-type lectin-like molecule-1 (CLL-1), a previously undescribed transmembrane glycoprotein. CLL-1 expression was analyzed with a human anti-CLL-1 antibody that was generated from the single chain Fv fragment. CLL-1 is restricted to the hematopoietic lineage, in particular to myeloid cells present in peripheral blood and bone marrow. CLL-1 is absent on uncommitted CD34+/CD38− or CD34+/CD33− stem cells and present on subsets of CD34+/CD38+ or CD34+/CD33+ progenitor cells. CLL-1 is not expressed in any other tissue. In contrast, analysis of primary AMLs demonstrated CLL-1 expression in 92% (68 of 74) of the samples. As an AML marker, CLL-1 was able to complement CD33, because 67% (8 of 12) of the CD33− AMLs expressed CLL-1. CLL-1 showed variable expression (10–60%) in CD34+ cells in chronic myelogenous leukemia and myelodysplastic syndrome but was absent in 12 of 13 cases of acute lymphoblastic leukemia. The AML reactivity combined with the restricted expression on normal cells identifies CLL-1 as a novel potential target for AML treatment.

First administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity.

Immediate passive immune prophylaxis as part of rabies post-exposure prophylaxis (PEP) often cannot be provided due to limited availability of human or equine rabies immunoglobulin (HRIG and ERIG, respectively). We report first clinical data from two phase I studies evaluating a monoclonal antibody cocktail CL184 against rabies. The studies included healthy adult subjects in the USA and India and involved two parts. First, subjects received a single intramuscular dose of CL184 or placebo in a double blind, randomized, dose-escalation trial. Second, open-label CL184 (20IU/kg) was co-administered with rabies vaccine. Safety was the primary objective and rabies virus neutralizing activity (RVNA) was investigated as efficacy parameter. Pain at the CL184 injection site was reported by less than 40% of subjects; no fever or local induration, redness or swelling was observed. RVNA was detectable from day 1 to day 21 after a single dose of CL184 20 or 40IU/kg. All subjects had adequate (>0.5IU/mL) RVNA levels from day 14 onwards when combined with rabies vaccine. CL184 appears promising as an alternative to RIG in PEP.

Novel human monoclonal antibody combination effectively neutralizing natural rabies virus variants and individual in vitro escape mutants

ABSTRACT The need to replace rabies immune globulin (RIG) as an essential component of rabies postexposure prophylaxis is widely acknowledged. We set out to discover a unique combination of human monoclonal antibodies (MAbs) able to replace RIG. Stringent criteria concerning neutralizing potency, affinity, breadth of neutralization, and coverage of natural rabies virus (RV) isolates and in vitro escape mutants were set for each individual antibody, and the complementarities of the two MAbs were defined at the onset. First, we identified and characterized one human MAb (CR57) with high in vitro and in vivo neutralizing potency and a broad neutralization spectrum. The linear antibody binding site was mapped on the RV glycoprotein as antigenic site I by characterizing CR57 escape mutants. Secondly, we selected using phage display a complementing antibody (CR4098) that recognized a distinct, nonoverlapping epitope (antigenic site III), showed similar neutralizing potency and breadth as CR57, and neutralized CR57 escape mutants. Reciprocally, CR57 neutralized RV variants escaping CR4098. Analysis of glycoprotein sequences of natural RV isolates revealed that the majority of strains contain both intact epitopes, and the few remaining strains contain at least one of the two. In vitro exposure of RV to the combination of CR57 and CR4098 yielded no escape mutants. In conclusion, a novel combination of human MAbs was discovered suitable to replace RIG.

Comparison of an Anti-Rabies Human Monoclonal Antibody Combination with Human Polyclonal Anti-Rabies Immune Globulin

The World Health Organization estimates human mortality from endemic canine rabies to be 55,000 deaths/year. Limited supply hampers the accessibility of appropriate lifesaving treatment, particularly in areas where rabies is endemic. Anti-rabies antibodies are key to protection against lethal rabies. Currently, only human and equine polyclonal anti-rabies immune globulin (HRIG and ERIG) is available. Replacement of HRIG and ERIG with a safer and more widely available product is recommended. We have recently identified a combination of 2 human monoclonal antibodies (MAbs), CR57 and CR4098, that has high potential. We here describe a head-to-head comparison between an CR57/CR4098 MAb cocktail and HRIG. The MAb cocktail neutralized all viruses from a panel of 26 representative street rabies virus isolates. In combination with vaccine, the MAb cocktail protected Syrian hamsters against lethal rabies when administered 24 h after exposure, comparable with the results obtained with HRIG. Furthermore, the MAb cocktail did not interfere with rabies vaccine differently from HRIG. These results demonstrate that the human MAb cocktail of CR57 and CR4098 is a safe and efficacious alternative to RIG in rabies postexposure prophylaxis.

The human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries.

Antibody phage display technology was used to identify human monoclonal antibodies that neutralize rabies virus (RV). A phage repertoire was constructed using antibody genes harvested from the blood of vaccinated donors. Selections using this repertoire and three different antigen formats of the RV glycoprotein (gp) resulted in the identification of 147 unique antibody fragments specific for the RV gp. Analysis of the DNA sequences of these antibodies demonstrated a large variation in the heavy‐ and light‐chain germ‐line gene usage, suggesting that a broad antibody repertoire was selected. The single‐chain variable fragment (scFv) antibodies were tested in vitro for RV neutralization, resulting in 39 specificities that neutralize the virus. Of the scFv clones, 21 were converted into full‐length human IgG1 format. Analysis of viral escape variants and binding competition experiments indicated that the majority of the neutralizing antibodies are directed against antigenic site III of the RV gp. The obtained specificities expand the set of human anti‐RV antibodies eligible for inclusion in an antibody cocktail aimed for use in rabies post‐exposure prophylaxis.

Molecular and Biological Characterization of Human Monoclonal Antibodies Binding to the Spike and Nucleocapsid Proteins of Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.

Novel Rabies Virus-Neutralizing Epitope Recognized by Human Monoclonal Antibody: Fine Mapping and Escape Mutant Analysis

ABSTRACT Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations.

Qualitative and Semiquantitative Analysis of Composite Mixtures of Antibodies by Native Mass Spectrometry

Native mass spectrometry was evaluated for the qualitative and semiquantitative analysis of composite mixtures of antibodies representing biopharmaceutical products coexpressed from single cells. We show that by using automated peak fitting of the ion signals in the native mass spectra, we can quantify the relative abundance of each of the antibodies present in mixtures, with an average accuracy of 3%, comparable to a cation exchange chromatography based approach performed in parallel. Moreover, using native mass spectrometry we were able to identify, separate, and quantify 9 antibodies present in a complex mixture of 10 antibodies, whereas this complexity could not be unraveled by cation exchange chromatography. Native mass spectrometry presents a valuable alternative to existing analytical methods for qualitative and semiquantitative profiling of biopharmaceutical products. It provides both the identity of each species in a mixture by mass determination and the relative abundance through comparison of relative ion signal intensities. Native mass spectrometry is a particularly effective tool for characterization of heterogeneous biopharmaceutical products such as bispecific antibodies and antibody mixtures.

Validation of the Rapid Fluorescent Focus Inhibition Test for Rabies Virus-Neutralizing Antibodies in Clinical Samples

ABSTRACT Monoclonal antibodies are successful biologics in treating a variety of diseases, including the prevention or treatment of viral infections. CL184 is a 1:1 combination of two human monoclonal IgG1 antibodies (CR57 and CR4098) against rabies virus, produced in the PER.C6 human cell line. The two antibodies are developed as replacements of human rabies immune globulin (HRIG) and equine rabies immune globulin (ERIG) in postexposure prophylaxis (PEP). The rapid fluorescent focus inhibition test (RFFIT) is a cell-based virus neutralization assay which is usually performed to determine the biological potency of a vaccine and to measure the levels of protection against rabies in humans and animals. In order to confirm the suitability of this assay as a pharmacodynamic assay, we conducted a validation using both HRIG- and CL184-spiked serum samples and sera from vaccinated donors. The validation results met all analytical acceptance criteria and showed that HRIG and CL184 serum concentrations can be compared. Stability experiments showed that serum samples were stable in various suboptimal conditions but that rabies virus should be handled swiftly once thawed. We concluded that the assay is suitable for the measurement of polyclonal and monoclonal rabies neutralizing antibodies in clinical serum samples.