NRBF2/Atg38 has been identified as the fifth subunit of the macroautophagic/autophagic class III phosphatidylinositol 3-kinase (PtdIns3K) complex, along with ATG14/Barkor, BECN1/Vps30, PIK3R4/p150/Vps15 and PIK3C3/Vps34. However, its functional mechanism and regulation are not fully understood. Here, we report that NRBF2 is a fine tuning regulator of PtdIns3K controlled by phosphorylation. Human NRBF2 is phosphorylated by MTORC1 at S113 and S120. Upon nutrient starvation or MTORC1 inhibition, NRBF2 phosphorylation is diminished. Phosphorylated NRBF2 preferentially interacts with PIK3C3/PIK3R4. Suppression of NRBF2 phosphorylation by MTORC1 inhibition alters its binding preference from PIK3C3/PIK3R4 to ATG14/BECN1, leading to increased autophagic PtdIns3K complex assembly, as well as enhancement of ULK1 protein complex association. Consequently, NRBF2 in its unphosphorylated form promotes PtdIns3K lipid kinase activity and autophagy flux, whereas its phosphorylated form blocks them. This study reveals NRBF2 as a critical molecular switch of PtdIns3K and autophagy activation, and its on/off state is precisely controlled by MTORC1 through phosphorylation.

Autophagy is a conserved eukaryotic pathway critical for cellular adaptation to changes in nutrition levels and stress. The class III phosphatidylinositol (PI)3-kinase complexes I and II (PI3KC3-C1 and -C2) are essential for autophagosome initiation and maturation, respectively, from highly curved vesicles. We used a cell-free reaction that reproduces a key autophagy initiation step, LC3 lipidation, as a biochemical readout to probe the role of autophagy-related gene (ATG)14, a PI3KC3-C1-specific subunit implicated in targeting the complex to autophagy initiation sites. We reconstituted LC3 lipidation with recombinant PI3KC3-C1, -C2, or various mutant derivatives added to extracts derived from a CRISPR/Cas9-generated ATG14-knockout cell line. Both complexes C1 and C2 require the C-terminal helix of VPS34 for activity on highly curved membranes. However, only complex C1 supports LC3 lipidation through the curvature-targeting amphipathic lipid packing sensor (ALPS) motif of ATG14. Furthermore, the ALPS motif and VPS34 catalytic activity are required for downstream recruitment of WD-repeat domain phosphoinositide-interacting protein (WIPI)2, a protein that binds phosphatidylinositol 3-phosphate and its product phosphatidylinositol 3, 5-bisphosphate, and a WIPI-binding protein, ATG2A, but do not affect membrane association of ATG3 and ATG16L1, enzymes contributing directly to LC3 lipidation. These data reveal the nuanced role of the ATG14 ALPS in membrane curvature sensing, suggesting that the ALPS has additional roles in supporting LC3 lipidation.

Autophagy is a fundamental cellular mechanism responsible for bulk turnover of cytoplasmic components. It is broadly related to many cellular activities, physiological processes, and pathological conditions. Autophagy entails a spatiotemporal interaction between cytosolic factors and membranes that are remodeled to encapsulate autophagic cargo within an autophagosome. Although majority of the factors [autophagy-related gene (Atg) proteins] involved in autophagy have been identified by genetic studies, the mechanism accounting for how these factors act upon the membrane to remodel it and efficiently recruit cargo for degradation is unclear. In vitro reconstitution of several different aspects of autophagy has provided important insights into the understanding of the mechanistic details underlying autophagic membrane remodeling and cargo recruitment. Here, we highlight these efforts toward studying autophagy through in vitro approaches.

There is a clinical need for new therapeutics to improve healing of chronic impaired wounds. Thus, we investigated how biopolymer conjugation could be used to improve the wound healing performance of a key growth factor for tissue regeneration: Sonic hedgehog (Shh). We generated two multivalent Shh conjugates (mvShh) using hyaluronic acid (HyA) with two different molecular weights (MWs), which exhibited equivalent potency and proteolytic protection in vitro. Using db/db diabetic mice, we showed that mvShh made with smaller HyA MW resulted in more rapid and robust neovascularization compared to mvShh made with larger MW HyA. Further, smaller mvShh conjugates resulted in faster wound resolution compared to the unconjugated Shh. This study is the first to show how the wound healing efficacy of multivalent protein-polymer conjugates is sensitive to the polymer MW, and our findings suggest that this parameter could be used to enhance the efficacy of growth factor conjugates.

Purpose: There is a critical need in immunotherapy drug development to enable focused and sustained immune cell modulation within a tumor to induce and propagate a system-wide anti-tumor response. We have developed a novel immunotherapy platform that could be used to generate geographically focused cancer cell growth inhibition or immune cell activation, thereby stimulating an anti-tumor immune response against primary solid tumors that can also travel to secondary metastases. Methods: Using published methods, we synthesized multivalent protein (MVP) conjugates by conjugating multiple copies (i.e. valency) of immune stimulating proteins, checkpoint inhibitors or anti-tumor antibodies to soluble, long-chain biopolymers. We verified that we can reproducibly generate MVP valencies ranging from 20-120 protein copies (±10%) per polymer backbone. We determined the binding affinity of these MVPs to their respective targets using biolayer interferometry and cell bioassays, and we measured the hydrodynamic radius of these immunotherapies using dynamic light scattering. Then, we injected fluorescently modified MVPs or their unconjugated counterparts directly into a variety of solid tumor models in mice. By taking longitudinal in vivo fluorescence measurements of the intratumoral (IT) drug signal over multiple days, we measured the IT half-life of each treatment. Results: Based on binding affinity measurements, we found that MVP potency increased directly with protein valency, and at high valency, the potency of MVPs were substantially greater than the unconjugated protein controls. Multivalent conjugation also increased the hydrodynamic radius of the MVPs to at least ten times larger than the unconjugated therapeutics. This large size was sufficient to slow the diffusion of MVP immunotherapies through dense tissues, such as solid tumors, as demonstrated by our in vivo studies. MVPs exhibited a higher IT drug signal with a more durable gradient within the tumor compared to the unconjugated controls, resulting in an extension of their IT half-lives by >5X in mouse solid tumors. Conclusions: The MVP platform can be used to modulate the potency and therapeutic durability for a wide range of immunotherapy targets. Further, the MVPs stay focused within the tumor after IT injection where they could generate a sustained anti-tumor immune response with minimal systemic exposure. Therefore, we expect MVP immunotherapies to have a better safety profile than IT or systemic delivery of an unconjugated therapeutic. We will continue to develop our internal MVP pipeline to finalize a candidate for IND-enabling studies. We are also seeking collaborations for co-development of additional immunotherapies that could benefit from the extended IT exposure and potency modulation enabled by the MVP platform. Citation Format: Livia Brier, Amy A. Twite, Adam Barnebey, Mavish Mahomed, Wesley M. Jackson. Using a multivalent immunotherapy platform to extend intratumoral therapeutic durability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4157.

Purpose: A key challenge in cancer drug development is optimizing the therapeutic index (TI), a measure of a treatment’s antitumor efficacy and its toxicity, such that the agent is effective in targeting the tumor with an acceptable safety profile. Despite the growing list of emerging treatment modalities, successful translation of a drug candidate into an effective therapeutic agent may be hampered by inability to dose the therapy within the TI. We have developed a novel platform for protein and/or antibody-based therapies by combining protein engineering with biopolymer physics to enhance antitumor mechanisms at lower treatment doses. Our long-term goal is to improve the TI of multiple therapeutic classes. Methods: A drug-engineering platform was developed based on multivalent protein (MVP) conjugates, wherein multiple copies (i.e., valency) of a therapeutic protein, such as monoclonal single-domain antibodies or interleukins, were covalently bound to soluble biopolymers, including heparin and modified celluloses (15 kDa-1.5 MDa). MVP valencies ranging from 2 to 200 protein copies (±10%) per polymer were generated, and the reproducibility of this process was verified by UV analysis and chromatography. The binding affinity of the MVPs to their targets was determined using biolayer interferometry and cell bioassays, and the hydrodynamic radius of the MVPs were measured using dynamic light scattering. Using longitudinal in vitro and in vivo fluorescence measurements of drug signal, the intratumoral (IT) retention, cellular internalization, and systemic distribution of each MVP was compared to that of equimolar unconjugated controls of the corresponding protein. Results: At high valency, the target binding affinity of MVPs was substantially greater than that of the unconjugated protein controls. MVPs with improved cell-receptor engagement could modulate the internalization/processing of antibody-drug conjugates. Multivalent conjugation also increased the hydrodynamic radius of the MVPs to >10-times larger than corresponding unconjugated proteins. This resulted in a high concentration of MVP therapeutics in solid tumors, as demonstrated by an extension of their IT half-lives by >5 times versus unconjugated controls in mouse models of multiple tumor types. Conclusions: An MVP platform can be applied to a wide range of therapeutic mechanisms. Our data demonstrate that MVP therapeutics can be localized within the tumor and potentially enable more potent tumor cell-killing responses compared with equimolar unconjugated protein controls. Thus, the MVP platform represents a compelling new tool for designing protein-based therapies. Further development of our MVP pipeline is continuing, with the aim of identifying a candidate for IND-enabling studies. Citation Format: Amy A. Twite, Adam Barnebey, Livia W. Brier, Brian Barnett, Wesley M. Jackson. A novel platform for protein engineering and modification to expand the therapeutic index of anti-tumor therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1525.

Purpose: The risk of severe side effects has limited the development of immune cell activating immunotherapies. There is a critical need in immunotherapy drug development to enable focused and sustained immune cell activation within a tumor to induce a system-wide anti-tumor response. We have developed a novel immunotherapy platform that could be used to generate geographically focused cancer cell growth inhibition or immune cell activation, thereby stimulating an anti-tumor immune response against primary solid tumors that can also travel to secondary metastases. Methods: Using published methods, we synthesized multivalent protein (MVP) conjugates by conjugating multiple copies (i.e. valency) of immune stimulating proteins (e.g. Interleukin-15) or anti-tumor antibodies (e.g. anti-Epidermal Growth Factor Receptor) to soluble, long-chain biopolymers (e.g. carboxymethylcellulose or hyaluronic acid, ~700 kDa). We verified that we can reproducibly generate MVP valencies ranging from 20-120 protein copies (±10%) per polymer backbone. We determined the binding affinity of these MVPs to their respective targets using biolayer interferometry and cell bioassays, and we measured the hydrodynamic radius of these immunotherapies using dynamic light scattering. Then, we injected flurosecently modified MVPs or their unconjugated counterparts directly into a variety of solid tumor models in mice. By taking longitudinal in vivo fuorescence measurments of the intratumoral (IT) drug signal over multiple days, we measured the IT half-life of each treatment. Results: Based on binding affinity measurements, we found that MVP potency increased directly with their protein valency, and at high valency, the potency of MVPs were substantially greater than the unconjugated protein controls. Multivalent conjugation also increased the hydrodynamic radius of the MVPs to at least ten times larger than the unconjugated therapeutics. This large size was sufficient to slow the diffusion of MVP immunotherapies through dense tissues, such as solid tumors, as demonstrated by our in vivo studies. MVPs exhibited a higher IT drug signal with a more durable gradient within the tumor compared to the unconjugated controls, resulting in an extension of their IT half-lives by >5X in mouse solid tumors. Conclusions: The MVP platform can be used to modulate the potency and therapeutic durability for a wide range of immunotherapy targets. Further, the MVPs stay focused within the tumor after IT injection where they could generate a sustained anti-tumor immune response with minimal systemic exposure. Therefore, we expect MVP immunotherapies to have a better safety profile than IT or systemic delivery of an unconjugated therapeutic. We will continue to develop our internal MVP pipeline to finalize a candidate for IND-enabling studies. We are also seeking to collaborations for co-development of additional immunotherapies that could benefit from the extended IT exposure and potency modulation enabled by the MVP platform. Citation Format: Livia W. Brier, Mavish Mahomed, Amy A. Twite, Adam Barnebey, Wesley M. Jackson. Extending intratumoral therapeutic durability using a multivalent immunotherapy platform [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P027.