"Stapling" scFv for multispecific biotherapeutics of superior properties.

Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation ("breathing") and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing.

Influenza vaccine-induced human bone marrow plasma cells decline within a year after vaccination.

A universal vaccine against influenza would ideally generate protective immune responses that are not only broadly reactive against multiple influenza strains but also long-lasting. Because long-term serum antibody levels are maintained by bone marrow plasma cells (BMPCs), we investigated the production and maintenance of these cells after influenza vaccination. We found increased numbers of influenza-specific BMPCs 4 weeks after immunization with the seasonal inactivated influenza vaccine, but numbers returned to near their prevaccination levels after 1 year.

A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene.

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5' untranslated region (UTR) of the Fragile X mental retardation-1 (FMR1) gene promoter. Conventionally, capillary electrophoresis fragment analysis on a genetic analyzer is used for the sizing of the CGG repeats of FMR1, but additional Southern blot analysis is required for exact measurement when the repeat number is higher than 200. Here, we present an accurate and robust polymerase chain reaction (PCR)-based method for quantification of the CGG repeats of FMR1.

Identification of balanced chromosomal rearrangements previously unknown among participants in the 1000 Genomes Project: implications for interpretation of structural variation in genomes and the future of clinical cytogenetics.

Purpose: Recent studies demonstrate that whole-genome sequencing enables detection of cryptic rearrangements in apparently balanced chromosomal rearrangements (also known as balanced chromosomal abnormalities, BCAs) previously identified by conventional cytogenetic methods. We aimed to assess our analytical tool for detecting BCAs in the 1000 Genomes Project without knowing which bands were affected.

Methods: The 1000 Genomes Project provides an unprecedented integrated map of structural variants in phenotypically normal subjects, but there is no information on potential inclusion of subjects with apparent BCAs akin to those traditionally detected in diagnostic cytogenetics laboratories.

Antibodies elicited by the first non-viral prophylactic cancer vaccine show tumor-specificity and immunotherapeutic potential.

MUC1 is a shared tumor antigen expressed on >80% of human cancers. We completed the first prophylactic cancer vaccine clinical trial based on a non-viral antigen, MUC1, in healthy individuals at-risk for colon cancer. This trial provided a unique source of potentially effective and safe immunotherapeutic drugs, fully-human antibodies affinity-matured in a healthy host to a tumor antigen.

Long-Lived Plasma Cells Are Contained within the CD19(-)CD38(hi)CD138(+) Subset in Human Bone Marrow.

Antibody responses to viral infections are sustained for decades by long-lived plasma cells (LLPCs). However, LLPCs have yet to be characterized in humans. Here we used CD19, CD38, and CD138 to identify four PC subsets in human bone marrow (BM).

Diversity, cellular origin and autoreactivity of antibody-secreting cell population expansions in acute systemic lupus erythematosus.

Acute systemic lupus erythematosus (SLE) courses with surges of antibody-secreting cells (ASCs) whose origin, diversity and contribution to serum autoantibodies remain unknown. Here, deep sequencing, proteomic profiling of autoantibodies and single-cell analysis demonstrated highly diversified ASCs punctuated by clones expressing the variable heavy-chain region VH4-34 that produced dominant serum autoantibodies. A fraction of ASC clones contained autoantibodies without mutation, a finding consistent with differentiation outside the germinal centers.

A proteomics approach for the identification and cloning of monoclonal antibodies from serum.

We describe a proteomics approach that identifies antigen-specific antibody sequences directly from circulating polyclonal antibodies in the serum of an immunized animal. The approach involves affinity purification of antibodies with high specific activity and then analyzing digested antibody fractions by nano-flow liquid chromatography coupled to tandem mass spectrometry. High-confidence peptide spectral matches of antibody variable regions are obtained by searching a reference database created by next-generation DNA sequencing of the B-cell immunoglobulin repertoire of the immunized animal.

Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer.

Purpose: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non-small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLC-associated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry.

Insertion of c-Myc into Igh induces B-cell and plasma-cell neoplasms in mice.

We used gene targeting in mice to insert a His(6)-tagged mouse c-Myc cDNA, Myc(His), head to head into the mouse immunoglobulin heavy-chain locus, Igh, just 5' of the intronic enhancer, Emu. The insertion of Myc(His) mimicked both the human t(8;14)(q24;q32) translocation that results in the activation of MYC in human endemic Burkitt lymphomas and the homologous mouse T(12;15) translocation that deregulates Myc in certain mouse plasmacytomas. Beginning at the age of 6 months, Myc(His) transgenic mice developed B-cell and plasma neoplasms, such as IgM(+) lymphoblastic B-cell lymphomas, Bcl-6(+) diffuse large B-cell lymphomas, and CD138(+) plasmacytomas, with an overall incidence of 68% by 21 months.

Novel targeted deregulation of c-Myc cooperates with Bcl-X(L) to cause plasma cell neoplasms in mice.

Deregulated expression of both Myc and Bcl-X(L) are consistent features of human plasma cell neoplasms (PCNs). To investigate whether targeted expression of Myc and Bcl-X(L) in mouse plasma cells might lead to an improved model of human PCN, we generated Myc transgenics by inserting a single-copy histidine-tagged mouse Myc gene, Myc(His), into the mouse Ig heavy-chain Calpha locus. We also generated Bcl-X(L) transgenic mice that contain a multicopy Flag-tagged mouse Bcl-x(Flag) transgene driven by the mouse Ig kappa light-chain 3' enhancer.