Dextramabs: A Novel Format of Antibody-Drug Conjugates Featuring a Multivalent Polysaccharide Scaffold.

Antibody-drug conjugates (ADCs) are multicomponent biomolecules that have emerged as a powerful tool for targeted tumor therapy. Combining specific binding of an immunoglobulin with toxic properties of a payload, they however often suffer from poor hydrophilicity when loaded with a high amount of toxins. To address these issues simultaneously, we developed dextramabs, a novel class of hybrid antibody-drug conjugates. In these architectures, the therapeutic antibody trastuzumab is equipped with a multivalent dextran polysaccharide that enables efficient loading with a potent toxin in a controllable fashion. Our modular chemoenzymatic approach provides an access to synthetic dextramabs bearing monomethyl auristatin as releasable cytotoxic cargo. They possess high drug-to-antibody ratios, remarkable hydrophilicity, and high toxicity in vitro.

Generation of Potent Anti-HER1/2 Immunotoxins by Protein Ligation Using Split Inteins.

Cell targeting protein toxins have gained increasing interest for cancer therapy aimed at increasing the therapeutic window and reducing systemic toxicity. Because recombinant expression of immunotoxins consisting of a receptor-binding and a cell-killing moiety is hampered by their high toxicity in a eukaryotic production host, most applications rely on recombinant production of fusion proteins consisting of an antibody fragment and a protein toxin in bacterial hosts such as Escherichia coli ( E. coli). These fusions often lack beneficial properties of whole antibodies like extended serum half-life or efficient endocytic uptake via receptor clustering. Here, we describe the production of full-length antibody immunotoxins using self-splicing split inteins. To this end, the short (11 amino acids) N-terminal intein part of the artificially designed split intein M86, a derivative of the Ssp DnaB intein, was recombinantly fused to the heavy chain of trastuzumab, a human epidermal growth factor receptor 2 (HER2) receptor targeting antibody and to a nanobody-Fc fusion targeting the HER1 receptor, respectively. Both antibodies were produced in Expi293F cells. The longer C-terminal counterpart of the intein was genetically fused to the protein toxins gelonin or Pseudomonas Exotoxin A, respectively, and expressed in E. coli via fusion to maltose binding protein. Using optimized in vitro splicing conditions, we were able to generate a set of specific and potent immunotoxins with IC50 values in the mid- to subpicomolar range.

Semi-synthetic vNAR libraries screened against therapeutic antibodies primarily deliver anti-idiotypic binders.

Anti-idiotypic binders which specifically recognize the variable region of monoclonal antibodies have proven to be robust tools for pharmacokinetic studies of antibody therapeutics and for the development of cancer vaccines. In the present investigation, we focused on the identification of anti-idiotypic, shark-derived IgNAR antibody variable domains (vNARs) targeting the therapeutic antibodies matuzumab and cetuximab for the purpose of developing specific capturing ligands. Using yeast surface display and semi-synthetic, CDR3-randomized libraries, we identified several highly specific binders targeting both therapeutic antibodies in their corresponding variable region, without applying any counter selections during screening. Importantly, anti-idiotypic vNAR binders were not cross-reactive towards cetuximab or matuzumab, respectively, and comprised good target recognition in the presence of human and mouse serum. When coupled to magnetic beads, anti-idiotypic vNAR variants could be used as efficient capturing tools. Moreover, a two-step procedure involving vNAR-functionalized beads was employed for the enrichment of potentially bispecific cetuximab × matuzumab antibody constructs. In conclusion, semi-synthetic and CDR3-randomized vNAR libraries in combination with yeast display enable the fast and facile identification of anti-idiotypic vNAR domains targeting monoclonal antibodies primarily in an anti-idiotypic manner.

A simplified procedure for antibody engineering by yeast surface display: Coupling display levels and target binding by ribosomal skipping.

Yeast surface display is a valuable, widely used method for protein engineering. However, current yeast display applications rely on the staining of epitope tags in order to verify full-length presentation of the protein of interest on the cell surface. We aimed at developing a modified yeast display approach that relies on ribosomal skipping, thereby enabling the translation of two proteins from one open reading frame and, in that manner, generating an intracellular fluorescence signal. This improved setup is based on a 2A sequence that is encoded between the protein to be displayed and a gene for green fluorescent protein (GFP). The intracellular GFP fluorescence signal of yeast cells correlates with full-length protein presentation and omits the need for the immunofluorescence detection of epitope tags. For method validation, shark-derived IgNAR variable domains (vNAR) were subjected to affinity maturation using the 2A-GFP system. Yeast library screening of full-length vNAR variants which were detected via GFP expression yielded the same high-affinity binder that had previously been isolated by our group using the conventional epitope tag-based display format. The presented method obviates the need for additional immunofluorescence cell staining, offering an easy and cost-friendly alternative to conventional epitope tag detections.

The Flow of the Gibbon LAVA Element Is Facilitated by the LINE-1 Retrotransposition Machinery.

LINE-Alu-VNTR-Alu-like (LAVA) elements comprise a family of non-autonomous, composite, non-LTR retrotransposons specific to gibbons and may have played a role in the evolution of this lineage. A full-length LAVA element consists of portions of repeats found in most primate genomes: CT-rich, Alu-like, and VNTR regions from the SVA retrotransposon, and portions of the AluSz and L1ME5 elements. To evaluate whether the gibbon genome currently harbors functional LAVA elements capable of mobilization by the endogenous LINE-1 (L1) protein machinery and which LAVA components are important for retrotransposition, we established a trans-mobilization assay in HeLa cells. Specifically, we tested if a full-length member of the older LAVA subfamily C that was isolated from the gibbon genome and named LAVAC, or its components, can be mobilized in the presence of the human L1 protein machinery. We show that L1 proteins mobilize the LAVAC element at frequencies exceeding processed pseudogene formation and human SVAE retrotransposition by > 100-fold and ≥3-fold, respectively. We find that only the SVA-derived portions confer activity, and truncation of the 3' L1ME5 portion increases retrotransposition rates by at least 100%. Tagged de novo insertions integrated into intronic regions in cell culture, recapitulating findings in the gibbon genome. Finally, we present alternative models for the rise of the LAVA retrotransposon in the gibbon lineage.