A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19(+) tumor cells.

To harness the potent tumor-killing capacity of T cells for the treatment of CD19(+) malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19(+) cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly target-dependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19(+) malignancies with an advantageous safety risk profile and anticipated dosing regimen.

RECRUIT-TandAbs: harnessing the immune system to kill cancer cells.

Tandem diabodies (TandAbs) are tetravalent bispecific molecules comprised of antibody variable domains with two binding sites for each antigen. RECRUIT-TandAbs can simultaneously engage an immune system effector cell, such as a natural killer cell or a cytotoxic T cell, and an antigen expressed specifically on a cancer cell, thus leading to killing of the cancer cell. Recruitment of immune effector cells is highly specific and mediated via binding of the TandAb to molecules expressed on the surface of these cells. Furthermore, the absence of an Fc domain allows TandAbs to avoid certain IgG-mediated side effects. With a molecular weight of approximately 110 kDa, TandAbs are far above the first-pass renal clearance limit, offering a pharmacokinetic advantage compared with smaller bispecific antibody formats. This article reviews the RECRUIT-TandAb technology and the therapeutic potential of these molecules.

A simple high-throughput purification method for hit identification in protein screening.

Phage and ribosome display technologies have emerged as important tools in the high-throughput screening of protein pharmaceuticals. However, a challenge created by the implementation of such tools is the need to purify large numbers of proteins for screening. While some assays may be compatible with crude bacterial lysates or periplasmic extracts, many functional assays, particularly cell-based assays, require protein of high purity and concentration. Here we evaluate several methods for small-scale, high-throughput protein purification. From our initial assessment we identified the HIS-Select 96-well filter plate system as the method of choice for further evaluation. This method was optimized and used to produce scFvs that were tested in cell-based functional assays. The behavior of HIS-Select purified scFvs in these assays was found to be similar to scFvs purified using a traditional large-scale 2-step purification method. The HIS-Select method allows high-throughput purification of hundreds of scFvs with yields in the 50-100 microg range, and of sufficient purity to allow evaluation in a cell-based proliferation assay. In addition, the use of a similar 96-well-based method facilitates the purification and subsequent screening of large numbers of IgGs and Fc fusion proteins generated through reformatting of scFv fragments.

MarA-mediated overexpression of the AcrAB efflux pump results in decreased susceptibility to tigecycline in Escherichia coli.

OBJECTIVES: The purpose of this study was to characterize decreased susceptibility to tigecycline in clinical isolates of Escherichia coli obtained during Phase 3 clinical trials. METHODS: Gene expression was analysed by transcriptional profile analysis and RT-PCR. Transposon mutagenesis with IS903kan was used for selection of transposon mutants. Transposon insertions were mapped by DNA sequencing and PCR analyses. The MICs were determined by broth microdilution. RESULTS: Both transcriptional profile analysis and Taqman RT-PCR demonstrated increased expression levels of MarA, a transcriptional activator, and AcrAB, an RND-type efflux pump, in the strains with elevated tigecycline MICs. Transposon mutagenesis generated nine mutants, the majority of which had either marA or acrB inactivated. Sequence analysis revealed a single nucleotide insertion in the open reading frame of the marR gene in less-susceptible strains of E. coli. CONCLUSIONS: This study suggested that a loss of MarR functionality due to a frameshift mutation resulted in constitutive overproduction of MarA and AcrAB and, consequently, in decreased susceptibility to tigecycline in clinical isolates of E. coli.

Overexpression of genes of the cell wall stimulon in clinical isolates of Staphylococcus aureus exhibiting vancomycin-intermediate- S. aureus-type resistance to vancomycin.

Custom-designed gene chips (Affymetrix) were used to determine genetic relatedness and gene expression profiles in Staphylococcus aureus isolates with increasing MICs of vancomycin that were recovered over a period of several weeks from the blood and heart valve of a patient undergoing extensive vancomycin therapy. The isolates were found to be isogenic as determined by the GeneChip based genotyping approach and thus represented a unique opportunity to study changes in gene expression that may contribute to the vancomycin resistance phenotype. No differences in gene expression were detected between the parent strain, JH1, and JH15, isolated from the nares of a patient contact. Few expression changes were observed between blood and heart valve isolates with identical vancomycin MICs. A large number of genes had altered expression in the late stage JH9 isolate (MIC = 8 microg/ml) compared to JH1 (MIC = 1 microg/ml). Most genes with altered expression were involved in housekeeping functions or cell wall biosynthesis and regulation. The sortase-encoding genes, srtA and srtB, as well as several surface protein-encoding genes were downregulated in JH9. Two hypothetical protein-encoding genes, SAS016 and SA2343, were dramatically overexpressed in JH9. Interestingly, 27 of the genes with altered expression in JH9 grown in drug-free medium were found to be also overexpressed when the parental strain JH1 was briefly exposed to inhibitory concentrations of vancomycin, and more than half (17 of 27) of the genes with altered expression belonged to determinants that were proposed to form part of a general cell wall stress stimulon (S. Utaida et al., Microbiology 149:2719-2732, 2003).

Use of ribotyping to retrospectively identify methicillin-resistant Staphylococcus aureus isolates from phase 3 clinical trials for tigecycline that are genotypically related to community-associated isolates.

A retrospective study was performed to identify methicillin-resistant Staphylococcus aureus (MRSA) isolates obtained from patients enrolled in phase 3 clinical trials for tigecycline that were genotypically similar to known community-associated MRSA (CA-MRSA) strains. The clinical trials were double-blind comparator studies for complicated skin and skin structure infections or complicated intra-abdominal infections. We obtained 85% of the MRSA isolates from patients with complicated skin and skin structure infections. Using ribotyping, MRSA isolates were compared with well-characterized North American CA-MRSA strains and negative-control hospital-associated (HA) MRSA strains by cluster analysis; 91 of the 173 isolates clustered with two groups of known CA-MRSA strains, 60% of which shared an indistinguishable ribotype. These isolates were subsequently tested for the presence of SCCmec type IV and the Panton-Valentine leukocidin (PVL)-encoding genes as well as susceptibility to clindamycin, characteristics that are typically associated with CA-MRSA; 89 of the 91 isolates carried the type IV SCCmec element and 76 were also positive for the PVL-encoding genes; 73 of these isolates were susceptible to clindamycin. A similar analysis performed on 26 nonclustering isolates identified only four with these characteristics; 89 of the 91 clustering isolates were inhibited by tigecycline at MICs of < or = 0.5 microg/ml. On the basis of clustering information and preliminary genetic characterization, it appears that ribotyping is a useful tool in identifying potential CA-MRSA isolates and 76 MRSA isolates from patients enrolled in the tigecycline phase 3 trials have genetic markers typically associated with CA-MRSA.

Multidrug resistance in Staphylococcus aureus due to overexpression of a novel multidrug and toxin extrusion (MATE) transport protein.

Efflux is an important mechanism of multidrug resistance (MDR) in bacteria. The multidrug and toxin extrusion (MATE) family is the most recently described group of MDR efflux proteins, none of which have previously been identified in Staphylococcus aureus. Two independently derived S. aureus mutants having efflux-related MDR phenotypes were studied using microarray technology and a marked overexpression of an open reading frame (ORF; mepA) encoding a protein homologous with MATE family proteins was observed in both. There was concomitant overexpression of ORFs in close proximity to mepA (approximately 100 bp) encoding a MarR-type regulator (mepR, upstream of mepA) and a protein of unknown function (mepB, downstream). Experiments in which mepA was overexpressed or disrupted revealed that the encoded protein has a broad substrate profile that includes several monovalent and divalent biocides and the fluoroquinolone antimicrobial agents norfloxacin and ciprofloxacin. The function of MepB is obscure, it does not contribute to the MDR phenotype conferred by MepA. MepR overexpression reversed the MDR phenotypes of both mutants by repressing mepA transcription. All three ORFs are preferentially transcribed as a single mepRAB unit, suggesting that the three genes form an operon.

A novel MATE family efflux pump contributes to the reduced susceptibility of laboratory-derived Staphylococcus aureus mutants to tigecycline.

Tigecycline, an expanded-broad-spectrum glycylcycline antibiotic is not affected by the classical tetracycline resistance determinants found in Staphylococcus aureus. The in vitro selection of mutants with reduced susceptibility to tigecycline was evaluated for two methicillin-resistant S. aureus strains by serial passage in increasing concentrations of tigecycline. Both strains showed a stepwise elevation in tigecycline MIC over a period of 16 days, resulting in an increase in tigecycline MIC of 16- and 32-fold for N315 and Mu3, respectively. Transcriptional profiling revealed that both mutants exhibited over 100-fold increased expression of a gene cluster, mepRAB (multidrug export protein), encoding a MarR-like transcriptional regulator (mepR), a novel MATE family efflux pump (mepA), and a hypothetical protein of unknown function (mepB). Sequencing of the mepR gene in the mutant strains identified changes that presumably inactivated the MepR protein, which suggested that MepR functions as a repressor of mepA. Overexpression of mepA in a wild-type background caused a decrease in susceptibility to tigecycline and other substrates for MATE-type efflux pumps, although it was not sufficient to confer high-level resistance to tigecycline. Complementation of the mepR defect by overexpressing a wild-type mepR gene reduced mepA transcription and lowered the tigecycline MIC in the mutants. Transcription of tet(M) also increased by over 40-fold in the Mu3 mutant. This was attributed to a deletion in the promoter region of the gene that removed a stem-loop responsible for transcriptional attenuation. However, overexpression of the tet(M) transcript in a tigecycline-susceptible strain was not enough to significantly increase the MIC of tigecycline. These results suggest that the overexpression of mepA but not tet(M) may contribute to decreased susceptibility of tigecycline in S. aureus.

Comparative genomics of Staphylococcus aureus musculoskeletal isolates.

Much of the research aimed at defining the pathogenesis of Staphylococcus aureus has been done with a limited number of strains, most notably the 8325-4 derivative RN6390. Several lines of evidence indicate that this strain is unique by comparison to clinical isolates of S. aureus. Based on this, we have focused our efforts on two clinical isolates (UAMS-1 and UAMS-601), both of which are hypervirulent in our animal models of musculoskeletal infection. In this study, we used comparative genomic hybridization to assess the genome content of these two isolates relative to RN6390 and each of seven sequenced S. aureus isolates. Our comparisons were done by using an amplicon-based microarray from the Pathogen Functional Genomics Resource Center and an Affymetrix GeneChip that collectively represent the genomes of all seven sequenced strains. Our results confirmed that UAMS-1 and UAMS-601 share specific attributes that distinguish them from RN6390. Potentially important differences included the presence of cna and the absence of isaB, sarT, sarU, and sasG in the UAMS isolates. Among the sequenced strains, the UAMS isolates were most closely related to the dominant European clone EMRSA-16. In contrast, RN6390, NCTC 8325, and COL formed a distinct cluster that, by comparison to the other four sequenced strains (Mu50, N315, MW2, and SANGER-476), was the most distantly related to the UAMS isolates and EMRSA-16.

Effect of mild acid on gene expression in Staphylococcus aureus.

During staphylococcal growth in glucose-supplemented medium, the pH of a culture starting near neutrality typically decreases by about 2 units due to the fermentation of glucose. Many species can comfortably tolerate the resulting mildly acidic conditions (pH, approximately 5.5) by mounting a cellular response, which serves to defend the intracellular pH and, in principle, to modify gene expression for optimal performance in a mildly acidic infection site. In this report, we show that changes in staphylococcal gene expression formerly thought to represent a glucose effect are largely the result of declining pH. We examine the cellular response to mild acid by microarray analysis and define the affected gene set as the mild acid stimulon. Many of the genes encoding extracellular virulence factors are affected, as are genes involved in regulation of virulence factor gene expression, transport of sugars and peptides, intermediary metabolism, and pH homeostasis. Key results are verified by gene fusion and Northern blot hybridization analyses. The results point to, but do not define, possible regulatory pathways by which the organism senses and responds to a pH stimulus.

Degradation of human antimicrobial peptide LL-37 by Staphylococcus aureus-derived proteinases.

Cathelicidin LL-37 is one of the few human bactericidal peptides with potent antistaphylococcal activity. In this study we examined the susceptibility of LL-37 to proteolytic degradation by two major proteinases produced by Staphylococcus aureus, a metalloproteinase (aureolysin) and a glutamylendopeptidase (V8 protease). We found that aureolysin cleaved and inactivated LL-37 in a time- and concentration-dependent manner. Analysis of the generated fragments by mass spectroscopy revealed that the initial cleavage of LL-37 by aureolysin occurred between the Arg19-Ile20, Arg23-Ile24, and Leu31-Val32 peptide bonds, instantly annihilating the antibacterial activity of LL-37. In contrast, the V8 proteinase hydrolyzed efficiently only the Glu16-Phe17 peptide bond, rendering the C-terminal fragment refractory to further degradation. This fragment (termed LL-17-37) displayed antibacterial activity against S. aureus at a molar level similar to that of the full-length LL-37 peptide, indicating that the antibacterial activity of LL-37 resides in the C-terminal region. In keeping with LL-37 degradation by aureolysin, S. aureus strains that produce significant amounts of this metalloprotease were found to be less susceptible to LL-17-37 than strains expressing no aureolysin activity. Taken together, these data suggest that aureolysin production by S. aureus contributes to the resistance of this pathogen to the innate immune system of humans mediated by LL-37.

Global gene expression in Staphylococcus aureus biofilms.

We previously demonstrated that mutation of the staphylococcal accessory regulator (sarA) in a clinical isolate of Staphylococcus aureus (UAMS-1) results in an impaired capacity to form a biofilm in vitro (K. E. Beenken, J. S. Blevins, and M. S. Smeltzer, Infect. Immun. 71:4206-4211, 2003). In this report, we used a murine model of catheter-based biofilm formation to demonstrate that a UAMS-1 sarA mutant also has a reduced capacity to form a biofilm in vivo. Surprisingly, mutation of the UAMS-1 ica locus had little impact on biofilm formation in vitro or in vivo. In an effort to identify additional loci that might be relevant to biofilm formation and/or the adaptive response required for persistence of S. aureus within a biofilm, we isolated total cellular RNA from UAMS-1 harvested from a biofilm grown in a flow cell and compared the transcriptional profile of this RNA to RNA isolated from both exponential- and stationary-phase planktonic cultures. Comparisons were done using a custom-made Affymetrix GeneChip representing the genomic complement of six strains of S. aureus (COL, N315, Mu50, NCTC 8325, EMRSA-16 [strain 252], and MSSA-476). The results confirm that the sessile lifestyle associated with persistence within a biofilm is distinct by comparison to the lifestyles of both the exponential and postexponential phases of planktonic culture. Indeed, we identified 48 genes in which expression was induced at least twofold in biofilms over expression under both planktonic conditions. Similarly, we identified 84 genes in which expression was repressed by a factor of at least 2 compared to expression under both planktonic conditions. A primary theme that emerged from the analysis of these genes is that persistence within a biofilm requires an adaptive response that limits the deleterious effects of the reduced pH associated with anaerobic growth conditions.

Analysis of mutations in the Staphylococcus aureus clfB promoter leading to increased expression.

A clfB : : tetK reporter was constructed in Staphylococcus aureus strains Newman and 8325-4, whereby the level of tetracycline resistance reflected the activity of the clfB promoter. Wild-type strains carrying a single copy of this construct exhibited a low level of tetracycline resistance, suggesting that the clfB promoter is weak. Spontaneous mutants that grew at higher tetracycline concentrations were isolated. Some were due to point mutations in the clfB promoter that led to increased expression of the tetK gene. The clfB promoter was identified by primer extension analysis and -35 and -10 elements were assigned. The promoter regions from the tetracycline-resistant mutants were sequenced and several had base changes within or adjacent to the -35 box. Three created the consensus -35 sequence TTGACA. The mutant clfB promoters were fused to lacZ. beta-Galactosidase activity was six- to ninefold higher in the mutant strains compared to the wild-type. The wild-type clfB gene was placed under the control of the mutant promoters. ClfB expression was higher than the corresponding wild-type strains and the protein was present on bacteria from the stationary phase instead of being confined to the exponential phase. Therefore, mutations in the clfB promoter that cause changes in the -35 region produce a stronger promoter that is capable of increased transcription and, as a result, increased expression of ClfB.

Clinical and epidemiological correlates of genotypes within the Mycobacterium avium complex defined by restriction and sequence analysis of hsp65.

Species identification of isolates of the Mycobacterium avium complex (MAC) remains a difficult task. Although M. avium and Mycobacterium intracellulare can be identified with expensive, commercially available probes, many MAC isolates remain unresolved, including those representing Mycobacterium lentiflavum as well as other potentially undefined species. PCR restriction analysis (PRA) of the hsp65 gene has been proposed as a rapid and inexpensive approach. We applied PRA to 278 MAC isolates, including 126 from blood of human immunodeficiency virus (HIV)-infected patients, 59 from sputum of HIV-negative patients with chronic obstructive pulmonary disease, 88 from environmental sources, and 5 pulmonary isolates from a different study. A total of 15 different PRA patterns were observed. For 27 representative isolates, a 441-bp fragment of the hsp65 gene was sequenced; based on 54 polymorphic sites, 18 different alleles were defined, including 12 alleles not previously reported. Species and phylogenetic relationships were more accurately defined by sequencing than by PRA or commercial probe. The distribution of PRA types and, by implication, phylogenetic lineages among blood isolates was significantly different from that for pulmonary and environmental isolates, suggesting that particular lineages have appreciably greater virulence and invasive potential.

Corneal pathogenesis of Staphylococcus aureus strain Newman.

PURPOSE: To determine the pathogenic role of gamma- and alpha-toxin in a rabbit model of Staphylococcus aureus keratitis. METHODS: S. aureus strains Newman (expressing gamma-toxin), Newman Delta(hlg) (deficient in gamma-toxin), Newman Delta(hlg)/pCU1 hlg(+) (chromosomal gamma-toxin-deficient mutant rescued by a plasmid encoding gamma-toxin), and Newman Delta(hla) (alpha-toxin-deficient) were intrastromally injected into rabbit corneas. Eyes were scored by slit lamp examination (SLE), and bacterial colony-forming units (CFU) per cornea were determined at 15, 20, and 25 hours after infection. Histologic examination of corneas was performed. Rabbits were immunized against alpha-toxin and subsequently challenged with S. aureus strain Newman. Western blot analyses of culture supernatants were performed to detect alpha-toxin production. RESULTS: All strains grew equivalently, producing approximately 7 log CFU per cornea at 25 hours after infection. SLE scores at 20 and 25 hours after infection revealed that strains Newman Delta(hlg) and Newman Delta(hla), although virulent, caused significantly less ocular damage and inflammation than their parent or the gamma-toxin genetically rescued strain (P