PSGL-1 Blockade Induces Classical Activation of Human Tumor-associated Macrophages.

The immune suppressive microenvironment is a major culprit for difficult-to-treat solid cancers. Particularly, inhibitory tumor-associated macrophages (TAM) define the resistant nature of the tumor milieu. To define tumor-enabling mechanisms of TAMs, we analyzed molecular clinical datasets correlating cell surface receptors with the TAM infiltrate. Though P-selectin glycoprotein ligand-1 (PSGL-1) is found on other immune cells and functions as an adhesion molecule, PSGL-1 is highly expressed on TAMs across multiple tumor types. siRNA-mediated knockdown and antibody-mediated inhibition revealed a role for PSGL-1 in maintaining an immune suppressed macrophage state. PSGL-1 knockdown or inhibition enhanced proinflammatory mediator release across assays and donors in vitro. In several syngeneic mouse models, PSGL-1 blockade alone and in combination with PD-1 blockade reduced tumor growth. Using a humanized tumor model, we observed the proinflammatory TAM switch following treatment with an anti-PSGL-1 antibody. In ex vivo patient-derived tumor cultures, a PSGL-1 blocking antibody increased expression of macrophage-derived proinflammatory cytokines, as well as IFNγ, indicative of T-cell activation. Our data demonstrate that PSGL-1 blockade reprograms TAMs, offering a new therapeutic avenue to patients not responding to T-cell immunotherapies, as well as patients with tumors devoid of T cells. SIGNIFICANCE: This work is a significant and actionable advance, as it offers a novel approach to treating patients with cancer who do not respond to T-cell checkpoint inhibitors, as well as to patients with tumors lacking T-cell infiltration. We expect that this mechanism will be applicable in multiple indications characterized by infiltration of TAMs.

L445P mutation on heavy chain stabilizes IgG4 under acidic conditions.

IgG4, a common type of therapeutic antibody, is less stable during manufacturing processes compared with IgG1. Aggregation and fragmentation are the two main challenges. Here, we report instability of the heavy chain (HC) C-terminal region under acidic conditions, which leads to cleavage and aggregation. Leu445, at the C-terminal region of the HC in IgG4, plays a critical role in its acid-induced fragmentation and subsequent aggregation. We found that mutating HC C-terminal Leu445 to Pro (the corresponding residue in IgG1) in IgG4_CDR-X significantly reduces fragmentation and aggregation, while mutating Pro445 to Leu in IgG1_CDR-X promotes fragmentation and aggregation. HC C-terminal Gly446 cleavage was observed in low pH citrate buffer and resulted in further fragmentation and aggregation, whereas, glycine buffer can completely inhibit the cleavage and aggregation. It is proposed that cleavages occur through acid-induced hydrolysis under acidic conditions and glycine stabilizes IgG4 via two main mechanisms: 1) product feedback inhibition of the hydrolysis reaction, and 2) stabilization of protein conformation by direct interaction with the peptide backbone and charged side chains. Experiments using IgG4 molecules IgG4_CDR-Y and IgG4_CDR-Z with the same CH domains as IgG4_CDR-X, but different complementarity-determining regions (CDRs), indicate that the stability of the HC C-terminal region is also closely related to the sequence of the CDRs. The stability of IgG4_CDR-X is significantly improved when binding to its target. Both observations suggest that there are potential interactions between Fab and CH2-CH3 domains, which could be the key factor affecting the stability of IgG antibodies.

Point mutations at the catalytic site of PCSK9 inhibit folding, autoprocessing, and interaction with the LDL receptor.

Circulating low-density lipoprotein cholesterol (LDLc) is regulated by membrane-bound LDL receptor (LDLr). Upon LDLc and LDLr interaction the complex is internalized by the cell, leading to LDLc degradation and LDLr recycling back to the cell surface. The proprotein convertase subtilisin/kexin type 9 (PCSK9) protein regulates this cycling. PCSK9 is secreted from the cell and binds LDLr. When the complex is internalized, PCSK9 prevents LDLr from shuttling back to the surface and instead targets it for degradation. PCSK9 is a serine protease expressed as a zymogen that undergoes autoproteolysis, though the two resulting protein domains remain stably associated as a heterodimer. This PCSK9 autoprocessing is required for the protein to be secreted from the cell. To date, direct analysis of PCSK9 autoprocessing has proven challenging, as no catalytically active zymogen has been isolated. A PCSK9 loss-of-function point mutation (Q152H) that reduces LDLc levels two-fold was identified in a patient population. LDLc reduction was attributed to a lack of PCSK9(Q152H) autoprocessing preventing secretion of the protein. We have isolated a zymogen form of PCSK9, PCSK9(Q152H), and a related mutation (Q152N), that can undergo slow autoproteolysis. We show that the point mutation prevents the formation of the mature form of PCSK9 by hindering folding, reducing the rate of autoproteolysis, and destabilizing the heterodimeric form of the protein. In addition, we show that the zymogen form of PCSK9 adopts a structure that is distinct from the processed form and is unable to bind a mimetic peptide based on the EGF-A domain of the LDLr.

A small molecule that binds and inhibits the ETV1 transcription factor oncoprotein.

Members of the ETS transcription factor family have been implicated in several cancers, where they are often dysregulated by genomic derangement. ETS variant 1 (ETV1) is an ETS factor gene that undergoes chromosomal translocation in prostate cancers and Ewing sarcomas, amplification in melanomas, and lineage dysregulation in gastrointestinal stromal tumors. Pharmacologic perturbation of ETV1 would be appealing in these cancers; however, oncogenic transcription factors are often deemed "undruggable" by conventional methods. Here, we used small-molecule microarray screens to identify and characterize drug-like compounds that modulate the biologic function of ETV1. We identified the 1,3,5-triazine small molecule BRD32048 as a top candidate ETV1 perturbagen. BRD32048 binds ETV1 directly, modulating both ETV1-mediated transcriptional activity and invasion of ETV1-driven cancer cells. Moreover, BRD32048 inhibits p300-dependent acetylation of ETV1, thereby promoting its degradation. These results point to a new avenue for pharmacologic ETV1 inhibition and may inform a general means to discover small molecule perturbagens of transcription factor oncoproteins.

N-glycosylation of enhanced aromatic sequons to increase glycoprotein stability.

N-glycosylation can increase the rate of protein folding, enhance thermodynamic stability, and slow protein unfolding; however, the molecular basis for these effects is incompletely understood. Without clear engineering guidelines, attempts to use N-glycosylation as an approach for stabilizing proteins have resulted in unpredictable energetic consequences. Here, we review the recent development of three "enhanced aromatic sequons," which appear to facilitate stabilizing native-state interactions between Phe, Asn-GlcNAc and Thr when placed in an appropriate reverse turn context. It has proven to be straightforward to engineer a stabilizing enhanced aromatic sequon into glycosylation-naïve proteins that have not evolved to optimize specific protein-carbohydrate interactions. Incorporating these enhanced aromatic sequons into appropriate reverse turn types within proteins should enhance the well-known pharmacokinetic benefits of N-glycosylation-based stabilization by lowering the population of protease-susceptible unfolded and aggregation-prone misfolded states, thereby making such proteins more useful in research and pharmaceutical applications.

Amyloid-ß forms fibrils by nucleated conformational conversion of oligomers.

Amyloid-ß amyloidogenesis is reported to occur via a nucleated polymerization mechanism. If this is true, the energetically unfavorable oligomeric nucleus should be very hard to detect. However, many laboratories have detected early nonfibrillar amyloid-ß oligomers without observing amyloid fibrils, suggesting that a mechanistic revision may be needed. Here we introduce Cys-Cys-amyloid-ß(1-40), which cannot bind to the latent fluorophore FlAsH as a monomer, but can bind FlAsH as an nonfibrillar oligomer or as a fibril, rendering the conjugates fluorescent. Through FlAsH monitoring of Cys-Cys-amyloid-ß(1-40) aggregation, we found that amyloid-ß(1-40) rapidly and efficiently forms spherical oligomers in vitro (85% yield) that are kinetically competent to slowly convert to amyloid fibrils by a nucleated conformational conversion mechanism. This methodology was used to show that plasmalogen ethanolamine vesicles eliminate the proteotoxicity-associated oligomerization phase of amyloid-ß amyloidogenesis while allowing fibril formation, rationalizing how low concentrations of plasmalogen ethanolamine in the brain are epidemiologically linked to Alzheimer's disease.

Protein native-state stabilization by placing aromatic side chains in N-glycosylated reverse turns.

N-glycosylation of eukaryotic proteins helps them fold and traverse the cellular secretory pathway and can increase their stability, although the molecular basis for stabilization is poorly understood. Glycosylation of proteins at naïve sites (ones that normally are not glycosylated) could be useful for therapeutic and research applications but currently results in unpredictable changes to protein stability. We show that placing a phenylalanine residue two or three positions before a glycosylated asparagine in distinct reverse turns facilitates stabilizing interactions between the aromatic side chain and the first N-acetylglucosamine of the glycan. Glycosylating this portable structural module, an enhanced aromatic sequon, in three different proteins stabilizes their native states by -0.7 to -2.0 kilocalories per mole and increases cellular glycosylation efficiency.

The core trisaccharide of an N-linked glycoprotein intrinsically accelerates folding and enhances stability.

The folding energetics of the mono-N-glycosylated adhesion domain of the human immune cell receptor cluster of differentiation 2 (hCD2ad) were studied systematically to understand the influence of the N-glycan on the folding energy landscape. Fully elaborated N-glycan structures accelerate folding by 4-fold and stabilize the beta-sandwich structure by 3.1 kcal/mol, relative to the nonglycosylated protein. The N-glycan's first saccharide unit accounts for the entire acceleration of folding and for 2/3 of the native state stabilization. The remaining third of the stabilization is derived from the next 2 saccharide units. Thus, the conserved N-linked triose core, ManGlcNAc(2), improves both the kinetics and the thermodynamics of protein folding. The native state stabilization and decreased activation barrier for folding conferred by N-glycosylation provide a powerful and potentially general mechanism for enhancing folding in the secretory pathway.