Anti-MrkA Monoclonal Antibodies Reveal Distinct Structural and Antigenic Features of MrkA.

Antibody therapy against antibiotics resistant Klebsiella pneumoniae infections represents a promising strategy, the success of which depends critically on the ability to identify appropriate antibody targets. Using a target-agnostic strategy, we recently discovered MrkA as a potential antibody target and vaccine antigen. Interestingly, the anti-MrkA monoclonal antibodies isolated through phage display and hybridoma platforms all recognize an overlapping epitope, which opens up important questions including whether monoclonal antibodies targeting different MrkA epitopes can be generated and if they possess different protective profiles. In this study we generated four anti-MrkA antibodies targeting different epitopes through phage library panning against recombinant MrkA protein. These anti-MrkA antibodies elicited strong in vitro and in vivo protections against a multi-drug resistant Klebsiella pneumoniae strain. Furthermore, mutational and epitope analysis suggest that the two cysteine residues may play essential roles in maintaining a MrkA structure that is highly compacted and exposes limited antibody binding/neutralizing epitopes. These results suggest the need for further in-depth understandings of the structure of MrkA, the role of MrkA in the pathogenesis of Klebsiella pneumoniae and the protective mechanism adopted by anti-MrkA antibodies to fully explore the potential of MrkA as an efficient therapeutic target and vaccine antigen

The Clinicopathological Features and Genetic Mutations in Gastric Cancer Patients According to EMAST and MSI Status

Background: There has been no report regarding the clinicopathological features and genetic mutations regarding elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in gastric cancer (GC). Methods: The correlation among EMAST status, microsatellite instability (MSI) status, mutations of common GC-related genes and 16 DNA repair-associated genes, and the clinicopathological features were analyzed. Results: Among the 360 GC patients enrolled, there were 76 (21.1%) with EMAST+ tumors and 284 with EMAST− tumors, and 59 (16.4%) were MSI-high (MSI-H) tumors, and 301 were microsatellite stable (MSS) tumors. Patients with EMAST+ tumors exhibited an earlier pathological T category and had more genetic mutations in the PI3K/AKT pathway, ARID1A and DNA repair-associated genes than those with EMAST− tumors. Patients with MSI-H tumors have more genetic mutations in the PI3K/AKT pathway and DNA repair-associated genes than those with MSS tumors. In the subgroup analysis for MSI-H GC, EMAST+ tumors were associated with earlier pathological T and N categories, earlier TNM stages, higher frequency of DNA-repair-associated genetic mutations, and a better survival rate than EMAST− tumors. Conclusions: PI3K/AKT pathway mutations may play an important role in EMAST+ and/or MSI-H GC. EMAST+/MSI-H tumors seem to represent a different subtype of gastric cancer from EMAST−/MSI-H tumors

Serotype-independent binding to KP strains by the new antibodies.

<p>Flow cytometry experiment was used to gauge the binding of the four new antibodies against three WT KP strains of different serotype as indicated. KP3 serves as the positive control and R347 is a human IgG₁ isotype control.</p

K_D measurement in IgG format against a mixture of monomeric and multimeric MrkA.

<p>K_D measurement in IgG format against a mixture of monomeric and multimeric MrkA.</p

Mutational analysis of MrkA and mAb characterizations by Western blot.

<p>3A. Western blot against full length MrkA and deletion mutants. Protein samples were resolved on a 4–12% SDS-PAGE gel under reducing condition and subjected to Western blot analysis by a murine anti-his mAb, KP3, and clones 1–6 IgGs as indicated underneath each blot. Sample arrangement is as follow: Lane 1, cell lysate from KP strain 43816DM; Lane 2, E.coli BL21 strain control; Lane 3–5 BL21 strain expressing his-tagged recombinant MrkA, MrkA with N terminal 40 amino acid deletion, and MrkA with C terminal 32 amino acid deletion, respectively. In the anti-his blot, a bracket indicates the positions of the monomeric, full length MrkA as well as the deletion mutants. 3B. Western blot analysis of various MrkA deletion mutants. MrkA deletion mutants 1 to 7 (del1-7) as indicated on the left were expressed in BL21 strain, resolved on a 4–12% SDS-PAGE gel under reducing condition, and subjected to Western blot detection by anti-his and KP3 antibodies as indicated. Lane arrangement: KP is the lysate prepared from 43816DM strain and E.coli represents lysate prepared from BL21 expressing the recombinant, his-tagged MrkA. 1–7 represent the mutants with corresponding numbers (del 1–7) described on the left. For both 3A and B, numbers to the left of the gel indicate the molecular weight in kDa.</p

Phage panning output screening cascade.

<p>More than 4000 colonies were picked for high throughput screening after phage panning, scFv.Fc conversion and transformation. Four clones including clones 1, 4, 5, and 6 were selected for further characterization.</p

Anti-MrkA antibody binding is influenced by the antigen presentation format.

<p>MrkA protein, either coated directly to the ELISA plate (left panel), or captured by streptavidin after biotinylation (right panel), was recognized differently by the anti-MrkA antibodies.</p