Bernd Wranik

Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non-Hodgkins lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non-Hodgkins lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for ADCs in humans.

A secreted protein microarray platform for extracellular protein interaction discovery

Characterization of the extracellular protein interactome has lagged far behind that of intracellular proteins, where mass spectrometry and yeast two-hybrid technologies have excelled. Improved methods for identifying receptor-ligand and extracellular matrix protein interactions will greatly accelerate biological discovery in cell signaling and cellular communication. These technologies must be able to identify low-affinity binding events that are often observed between membrane-bound coreceptor molecules during cell-cell or cell-extracellular matrix contact. Here we demonstrate that functional protein microarrays are particularly well-suited for high-throughput screening of extracellular protein interactions. To evaluate the performance of the platform, we screened a set of 89 immunoglobulin (Ig)-type receptors against a highly diverse extracellular protein microarray with 686 genes represented. To enhance detection of low-affinity interactions, we developed a rapid method to assemble bait Fc fusion proteins into multivalent complexes using protein A microbeads. Based on these screens, we developed a statistical methodology for hit calling and identification of nonspecific interactions on protein microarrays. We found that the Ig receptor interactions identified using our methodology are highly specific and display minimal off-target binding, resulting in a 70% true-positive to false-positive hit ratio. We anticipate that these methods will be useful for a wide variety of functional protein microarray users.

Defining the Ligand Specificity of the Deleted in Colorectal Cancer (DCC) Receptor

The growth and guidance of many axons in the developing nervous system require Netrin-mediated activation of Deleted in Colorectal Cancer (DCC) and other still unknown signaling cues. Commissural axon guidance defects are more severe in DCC mutant mice than Netrin-1 mutant mice, suggesting additional DCC activating signals besides Netrin-1 are involved in proper axon growth. Here we report that interaction screens on extracellular protein microarrays representing over 1,000 proteins uniquely identified Cerebellin 4 (CBLN4), a member of the C1q-tumor necrosis factor (TNF) family, and Netrin-1 as extracellular DCC-binding partners. Immunofluorescence and radio-ligand binding studies demonstrate that Netrin-1 competes with CBLN4 binding at an overlapping site within the membrane-proximal fibronectin domains (FN) 4–6 of DCC and binds with ∼5-fold higher affinity. CBLN4 also binds to the DCC homolog, Neogenin-1 (NEO1), but with a lower affinity compared to DCC. CBLN4-null mice did not show a defect in commissural axons of the developing spinal cord but did display a transient increase in the number of wandering axons in the brachial plexus, consistent with a role in axon guidance. Overall, the data solidifies CBLN4 as a bona fide DCC ligand and strengthens its implication in axon guidance.

LUZ-Y, a Novel Platform for the Mammalian Cell Production of Full-length IgG-bispecific Antibodies

Background: Bispecific antibodies (bisAbs) enable novel therapeutic strategies by concomitantly binding two unique antigens. Results: IgG-like bisAbs can be produced in mammalian cells through the use of cleavable leucine zippers and GGS tethers. Conclusion: The LUZ-Y platform enables mammalian cell production of bisAbs. Significance: bisAbs structurally similar to full-length hIgG should enable novel therapies with minimal risk of adverse immune response. The ability of bispecific antibodies to simultaneously bind two unique antigens has great clinical potential. However, most approaches utilized to generate bispecific antibodies yield antibody-like structures that diverge significantly from the structure of archetype human IgG, and those that do approach structural similarity to native antibodies are often challenging to engineer and manufacture. Here, we present a novel platform for the mammalian cell production of bispecific antibodies that differ from their parental mAbs by only a single point mutation per heavy chain. Central to this platform is the addition of a leucine zipper to the C terminus of the CH3 domain of the antibody that is sufficient to drive the heterodimeric assembly of antibody heavy chains and can be readily removed post-purification. Using this approach, we developed various antibody constructs including one-armed Abs, bispecific antibodies that utilize a common light chain, and bispecific antibodies that pair light chains to their cognate heavy chains via peptide tethers. We have applied this technology to various antibody pairings and will demonstrate the engineering, purification, and biological activity of these antibodies herein.

The extracellular interactome of the human adenovirus family reveals diverse strategies for immunomodulation.

Viruses encode secreted and cell-surface expressed proteins essential to modulate host immune defenses and establish productive infections. However, to date there has been no systematic study of the extracellular interactome of any human virus. Here we utilize the E3 proteins, diverse and rapidly evolving transmembrane-containing proteins encoded by human adenoviruses, as a model system to survey the extracellular immunomodulatory landscape. From a large-scale protein interaction screen against a microarray of more than 1,500 human proteins, we find and validate 51 previously unidentified virus–host interactions. Our results uncover conserved strategies as well as substantial diversity and multifunctionality in host targeting within and between viral species. Prominent modulation of the leukocyte immunoglobulin-like and signalling lymphocyte activation molecule families and a number of inhibitory receptors were identified as hubs for viral perturbation, suggesting unrecognized immunoregulatory strategies. We describe a virus–host extracellular interaction map of unprecedented scale that provides new insights into viral immunomodulation.