Targeted chain-exchange-mediated reconstitution of a split type-I cytokine for conditional immunotherapy.

Antibody-cytokine fusions targeted against tumor-associated antigens (TAAs) are promising cancer immunotherapy agents, with many such molecules currently undergoing clinical trials. However, due to the limited number of tumor-specific targets, on-target off-tumor effects can lead to systemic toxicity. Additionally, targeted cytokines can be scavenged by cytokine receptors on peripheral cells, decreasing tumor penetration. This study aims at overcoming these issues by engineering a platform for targeted conditionally active type I cytokines. Building on our previously reported PACE (Prodrug-Activating Chain Exchange) platform, we split the type I cytokine interleukin-4 (IL-4) to create two inactive IL-4 prodrugs, and fused these split IL-4 counterparts to the C-termini of antibody-like molecules that undergo proximity-induced chain exchange. In doing so, we developed IL-4 prodrugs that preferentially reconstitute into active IL-4 on target cells. We demonstrate that pre-assembled split IL-4 (without additional inactivation) retains activity and present two different strategies of splitting and inactivating IL-4. Using an IL-4 responsive cell-line, we show that IL-4 prodrugs are targeted to TAAs on target cells and regain activity upon chain exchange, primarily in a cis-activation setting. Furthermore, we demonstrate that split IL-4 complementation is also possible in a trans-activation setting, which opens up the possibility for activation of immune cells in the tumor vicinity. We demonstrate that targeted on-cell prodrug conversion is more efficient than nonspecific activation in-solution. Due to the structural similarity between IL-4 and other type I cytokines relevant in cancer immunotherapy such as IL-2, IL-15, and IL-21, cytokine-PACE may be expanded to develop a variety of targeted conditionally active cytokines for cancer immunotherapy.

Prodrug-Activating Chain Exchange (PACE) converts targeted prodrug derivatives to functional bi- or multispecific antibodies.

Driven by the potential to broaden the target space of conventional monospecific antibodies, the field of multi-specific antibody derivatives is growing rapidly. The production and screening of these artificial proteins entails a high combinatorial complexity. Antibody-domain exchange was previously shown to be a versatile strategy to produce bispecific antibodies in a robust and efficient manner. Here, we show that the domain exchange reaction to generate hybrid antibodies also functions under physiological conditions. Accordingly, we modified the exchange partners for use in therapeutic applications, in which two inactive prodrugs convert into a product with additional functionalities. We exemplarily show the feasibility for generating active T cell bispecific antibodies from two inactive prodrugs, which per se do not activate T cells alone. The two complementary prodrugs harbor antigen-targeting Fabs and non-functional anti-CD3 Fvs fused to IgG-CH3 domains engineered to drive chain-exchange reactions between them. Importantly, Prodrug-Activating Chain Exchange (PACE) could be an attractive option to conditionally activate therapeutics at the target site. Several examples are provided that demonstrate the efficacy of PACE as a new principle of cancer immunotherapy in vitro and in a human xenograft model.

Doa10 is a membrane protein retrotranslocase in ER-associated protein degradation.

In endoplasmic reticulum-associated protein degradation (ERAD), membrane proteins are ubiquitinated, extracted from the membrane, and degraded by the proteasome. The cytosolic ATPase Cdc48 drives extraction by pulling on polyubiquitinated substrates. How hydrophobic transmembrane (TM) segments are moved from the phospholipid bilayer into cytosol, often together with hydrophilic and folded ER luminal protein parts, is not known. Using a reconstituted system with purified proteins from Saccharomyces cerevisiae, we show that the ubiquitin ligase Doa10 (Teb-4/MARCH6 in animals) is a retrotranslocase that facilitates membrane protein extraction. A substrate's TM segment interacts with the membrane-embedded domain of Doa10 and then passively moves into the aqueous phase. Luminal substrate segments cross the membrane in an unfolded state. Their unfolding occurs on the luminal side of the membrane by cytoplasmic Cdc48 action. Our results reveal how a membrane-bound retrotranslocase cooperates with the Cdc48 ATPase in membrane protein extraction.

The inside of a cell contains many different compartments called organelles, which are separated by membranes. Each organelle is composed of a unique set of proteins and performs specific roles in the cell. The endoplasmic reticulum, or ER for short, is an organelle where many proteins are produced. Most of these proteins are then released from the cell or sorted to other organelles. The ER has a strict quality control system that ensures any faulty proteins are quickly marked for the cell to destroy. However, the destruction process itself does not happen in the ER, so faulty proteins first need to leave this organelle. This is achieved by a group of proteins known as endoplasmic reticulum-associated protein degradation machinery (or ERAD for short). To extract a faulty protein from the ER, proteins of the ER and outside the ER cooperate. First, an ERAD protein called Doa10 attaches a small protein tag called ubiquitin to the faulty proteins to mark them for destruction. Then, outside of the ER, a protein called Cdc48 'grabs' the ubiquitin tag and pulls. But that is only part of the story. Many of the proteins made by the ER have tethers that anchor them firmly to the membrane, making them much harder to remove. To get a better idea of how the extraction works, Schmidt et al. rebuilt the ERAD machinery in a test tube. This involved purifying proteins from yeast and inserting them into artificial membranes, allowing closer study of each part of the process. This revealed that attaching ubiquitin tags to faulty proteins is only one part of Doa10's role; it also participates in the extraction itself. Part of Doa10 resides within the membrane, and this 'membrane-spanning domain' can interact with faulty proteins, loosening their membrane anchors. At the same time, Cdc48 pulls from the outside. This pulling force causes the faulty proteins to unfold, allowing them to pass through the membrane. Given these findings, the next step is to find out exactly how Doa10 works by looking at its three-dimensional structure. This could have implications not only for the study of ERAD, but of similar quality control processes in other organelles too. A build-up of faulty proteins can cause diseases like neurodegeneration, so understanding how cells remove faulty proteins could help future medical research.

Hrd1 forms the retrotranslocation pore regulated by auto-ubiquitination and binding of misfolded proteins.

During endoplasmic-reticulum-associated protein degradation (ERAD), misfolded proteins are polyubiquitinated, extracted from the ER membrane and degraded by the proteasome1-4. In a process called retrotranslocation, misfolded luminal proteins first need to traverse the ER membrane before ubiquitination can occur in the cytosol. It was suggested that the membrane-embedded ubiquitin ligase Hrd1 forms a retrotranslocation pore regulated by cycles of auto- and deubiquitination5-8. However, the mechanism by which auto-ubiquitination affects Hrd1 and allows polypeptides to cross the membrane and whether Hrd1 forms a membrane-spanning pore remained unknown. Here, using purified Hrd1 incorporated into different model membranes, we show that Hrd1 auto-ubiquitination leads to the opening of a pore. Substrate binding increases the pore size and its activity, whereas deubiquitination closes the pore and renders it unresponsive to substrate. We identify two binding sites for misfolded proteins in Hrd1, a low-affinity luminal site and a high-affinity cytoplasmic site formed following auto-ubiquitination of specific lysine residues in Hrd1's RING domain. We propose that the affinity difference between the luminal and cytoplasmic binding sites provides the initial driving force for substrate movement through Hrd1.

Biofortified orange maize enhances ß-cryptoxanthin concentrations in egg yolks of laying hens better than tangerine peel fortificant.

The xanthophyll ß-cryptoxanthin provides vitamin A and has other purported health benefits. Laying hens deposit xanthophyll carotenoids into egg yolk. Hens (n = 8/group) were fed conventional-bred high ß-cryptoxanthin biofortified (orange) maize, tangerine peel-fortified white maize, lutein-fortified yellow maize, or white maize for 40 d to investigate yolk color changes using L*a*b* scales, yolk carotenoid enhancement, and hen vitamin A status. Yolks from hens fed orange maize had scores indicating a darker, orange color and mean higher ß-cryptoxanthin, zeaxanthin, and ß-carotene concentrations (8.43 ± 1.82, 23.1 ± 4.8, 0.16 ± 0.08 nmol/g, respectively) than other treatments (P < 0.0001). Yolk retinol concentrations (mean: 14.4 ± 3.42 nmol/g) were similar among groups and decreased with time (P < 0.0001). Hens fed orange maize had higher liver retinol (0.53 ± 0.20 µmol/g liver) than other groups (P < 0.0001). ß-Cryptoxanthin-biofortified eggs could be another choice for consumers, providing enhanced color through a provitamin A carotenoid and supporting eggs' status as a functional food.