Protein phosphorylation is a prevalent translational modification, and its dysregulation has been implicated in various diseases, including cancer. Despite its significance, there is a lack of specific inhibitors of the FCP/SCP-type Ser/Thr protein phosphatase Scp1, characterized by high specificity and affinity. In this study, we focused on adnectin, an antibody-mimetic protein, aiming to identify Scp1-specific binding molecules with a broad binding surface that target the substrate-recognition site of Scp1. Biopanning of Scp1 was performed using an adnectin-presenting phage library with a randomized FG loop. We succeeded in identifying FG-1Adn, which showed high affinity and specificity for Scp1. Ala scanning analysis of the Scp1-binding sequence in relation to the FG-1 peptide revealed that hydrophobic residues, including aromatic amino acids, play important roles in Scp1 recognition. Furthermore, FG-1Adn was found to co-localize with Scp1 in cells, especially on the plasma membrane. In addition, Western blotting analysis showed that FG-1Adn increased the phosphorylation level of the target protein of Scp1 in cells, indicating that FG-1Adn can inhibit the function of Scp1. These results suggest that FG-1Adn can be used as a specific inhibitor of Scp1.

BACKGROUND: Adnectin is a protein family derived from the 10th type III domain of human fibronectin (10Fn3) with high-affinity targeting capabilities. Positron emission tomography (PET) probes derived from anti-programmed death ligand-1 (PD-L1) Adnectins, including 18F- and 68Ga-labeled BMS-986192, are recently developed for the prediction of patient response to immune checkpoint blockade. The 68Ga-labeled BMS-986192, in particular, is an attractive probe for under-developed regions due to the broader availability of 68Ga. However, the pharmacokinetics and biocompatibility of 68Ga-labeled BMS-986192 are still unknown, especially in non-human primates, impeding its further clinical translation.
METHODS: We developed a variant of 68Ga-labeled BMS-986192 using 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) as the radionuclide-chelator. The resultant probe, 68Ga-NODAGA-BMS986192, was evaluated in terms of targeting specificity using a bilateral mouse tumor model inoculated with wild-type B16F10 and B16F10 transduced with human PD-L1 (hPD-L1-B16F10). The dynamic biodistribution and radiation dosimetry of this probe were also investigated in non-human primate cynomolgus.
RESULTS: 68Ga-NODAGA-BMS986192 was prepared with a radiochemical purity above 99%. PET imaging with 68Ga-NODAGA-BMS986192 efficiently delineated the hPD-L1-B16F10 tumor at 1 h post-injection. The PD-L1-targeting capability of this probe was further confirmed using in vivo blocking assay and ex vivo biodistribution studies. PET dynamic imaging in both mouse and cynomolgus models revealed a rapid clearance of the probe via the renal route, which corresponded to the low background signals of the PET images. The probe also exhibited a favorable radiation dosimetry profile with a total-body effective dose of 6.34E-03 mSv/MBq in male cynomolgus.
CONCLUSIONS: 68Ga-NODAGA-BMS986192 was a feasible and safe tool for the visualization of human PD-L1. Our study also provided valuable information on the potential of targeted PET imaging using Adnectin-based probes.

GSK3732394 is a multi-specific biologic inhibitor of HIV entry currently under clinical evaluation. A key component of this molecule is an adnectin (6940_B01) that binds to CD4 and inhibits downstream actions of gp160. Studies were performed to determine the binding site of the adnectin on CD4 and to understand the mechanism of inhibition. Using hydrogen-deuterium exchange with mass spectrometry (HDX), CD4 peptides showed differential rates of deuteration (either enhanced or slowed) in the presence of the adnectin that mapped predominantly to the interface of domains 2 and 3 (D2-D3). In addition, an X-ray crystal structure of an ibalizumab Fab/CD4(D1-D4)/adnectin complex revealed an extensive interface between the adnectin and residues on CD4 domains D2-D4 that stabilize a novel T-shaped CD4 conformation. A cryo-EM map of the gp140/CD4/GSK3732394 complex clearly shows the bent conformation for CD4 while bound to gp140. Mutagenic analyses on CD4 confirmed that amino acid F202 forms a key interaction with the adnectin. In addition, amino acid L151 was shown to be a critical indirect determinant of the specificity for binding to the human CD4 protein over related primate CD4 molecules, as it appears to modulate CD4\'s flexibility to adopt the adnectin-bound conformation. The significant conformational change of CD4 upon adnectin binding brings the D1 domain of CD4 in proximity to the host cell membrane surface, thereby re-orienting the gp120 binding site in a direction that is inaccessible to incoming virus due to a steric clash between gp160 trimers on the virus surface and the target cell membrane.

The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

As a non-antibody scaffold, monobodies based on the fibronectin type III (FN3) domain overcome antibody size and complexity while maintaining analogous binding loops. However, antibodies and their derivatives remain the gold standard for the design of new therapeutics. In response, clinical-stage therapeutic proteins based on the FN3 domain are beginning to use native fibronectin function as a point of differentiation. The small and simple structure of monomeric monobodies confers increased tissue distribution and reduced half-life, whilst the absence of disulphide bonds improves stability in cytosolic environments. Where multi-specificity is challenging with an antibody format that is prone to mis-pairing between chains, multiple FN3 domains in the fibronectin assembly already interact with a large number of molecules. As such, multiple monobodies engineered for interaction with therapeutic targets are being combined in a similar beads-on-a-string assembly which improves both efficacy and pharmacokinetics. Furthermore, full length fibronectin is able to fold into multiple conformations as part of its natural function and a greater understanding of how mechanical forces allow for the transition between states will lead to advanced applications that truly differentiate the FN3 domain as a therapeutic scaffold.

The concept of engineering robust protein scaffolds for novel binding functions emerged 20 years ago, one decade after the advent of recombinant antibody technology. Early examples were the Affibody, Monobody (Adnectin), and Anticalin proteins, which were derived from fragments of streptococcal protein A, from the tenth type III domain of human fibronectin, and from natural lipocalin proteins, respectively. Since then, this concept has expanded considerably, including many other protein templates. In fact, engineered protein scaffolds with useful binding specificities, mostly directed against targets of biomedical relevance, constitute an area of active research today, which has yielded versatile reagents as laboratory tools. However, despite strong interest from basic science, only a handful of those protein scaffolds have undergone biopharmaceutical development up to the clinical stage. This includes the abovementioned pioneering examples as well as designed ankyrin repeat proteins (DARPins). Here we review the current state and clinical validation of these next-generation therapeutics.

Protein aggregation is a phenomenon that has attracted considerable attention within the pharmaceutical industry from both a developability standpoint (to ensure stability of protein formulations) and from a research perspective for neurodegenerative diseases. Experimental identification of aggregation behavior in proteins can be expensive; and hence, the development of accurate computational approaches is crucial. The existing methods for predicting protein aggregation rely mostly on the primary sequence and are typically trained on amyloid-like proteins. However, the training bias toward beta amyloid peptides may worsen prediction accuracy of such models when applied to larger protein systems. Here, we present a novel algorithm to identify aggregation-prone regions in proteins termed \"AggScore\" that is based entirely on three-dimensional structure input. The method uses the distribution of hydrophobic and electrostatic patches on the surface of the protein, factoring in the intensity and relative orientation of the respective surface patches into an aggregation propensity function that has been trained on a benchmark set of 31 adnectin proteins. AggScore can accurately identify aggregation-prone regions in several well-studied proteins and also reliably predict changes in aggregation behavior upon residue mutation. The method is agnostic to an amyloid-specific aggregation context and thus may be applied to globular proteins, small peptides and antibodies.

The N17 region of gp41 in HIV-1 is the most conserved region in gp160. mRNA selection technologies were used to identify an adnectin that binds to this region and inhibits gp41-induced membrane fusion. Additional selection conditions were used to optimize the adnectin to greater potency (5.4 ± 2.6 nM) against HIV-1 and improved binding affinity for an N17-containing helical trimer (0.8 ± 0.4 nM). Resistance to this adnectin mapped to a single Glu-to-Arg change within the N17 coding region. The optimized adnectin (6200_A08) exhibited high potency and broad-spectrum activity against 123 envelope proteins and multiple clinical virus isolates, although certain envelope proteins did exhibit reduced susceptibility to 6200_A08 alone. The reduced potency could not be correlated with sequence changes in the target region and was thought to be the result of faster kinetics of fusion mediated by these envelope proteins. Optimized linkage of 6200_A08 with a previously characterized adnectin targeting CD4 produced a highly synergistic molecule, with the potency of the tandem molecule measured at 37 ± 1 pM. In addition, these tandem molecules now exhibited few potency differences against the same panel of envelope proteins with reduced susceptibility to 6200_A08 alone, providing evidence that they did not have intrinsic resistance to 6200_A08 and that coupling 6200_A08 with the anti-CD4 adnectin may provide a higher effective on rate for gp41 target engagement.IMPORTANCE There continue to be significant unmet medical needs for patients with HIV-1 infection. One way to improve adherence and decrease the likelihood of drug-drug interactions in HIV-1-infected patients is through the development of long-acting biologic inhibitors. This study describes the development and properties of an adnectin molecule that targets the most conserved region of the gp41 protein and inhibits HIV-1 with good potency. Moreover, when fused to a similar adnectin targeted to the human CD4 protein, the receptor for HIV-1, significant synergies in potency and efficacy are observed. These inhibitors are part of an effort to develop a larger biologic molecule that functions as a long-acting self-administered regimen for patients with HIV-1 infection.

Higher-order structure (HOS) characterization of therapeutic protein-drug conjugates for comprehensive assessment of conjugation-induced protein conformational changes is an important consideration in the biopharmaceutical industry to ensure proper behavior of protein therapeutics. In this study, conformational dynamics of a small therapeutic protein, adnectin 1, together with its drug conjugate were characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS) with different spatial resolutions. Top-down HDX allows detailed assessment of the residue-level deuterium content in the payload conjugation region. HDX-MS dataset revealed the ability of peptide-based payload/linker to retain deuterium in HDX experiments. Combined results from intact, top-down, and bottom-up HDX indicated no significant conformational changes of adnectin 1 upon payload conjugation. Graphical Abstract ᅟ.

Although chimeric antigen receptor (CAR)-engineered T cell therapy has achieved encouraging clinical trial results for treating hematological cancers, further optimization can likely expand this therapeutic success to more patients and other cancer types. Most CAR constructs used in clinical trials incorporate single chain variable fragment (scFv) as the extracellular antigen recognition domain. The immunogenicity of nonhuman scFv could cause host rejection against CAR T cells and compromise their persistence and efficacy. The limited availability of scFvs and slow discovery of new monoclonal antibodies also limit the development of novel CAR constructs. Adnectin, a class of affinity molecules derived from the tenth type III domain of human fibronectin, can be an alternative to scFv as an antigen-binding moiety in the design of CAR molecules. We constructed adnectin-based CARs targeting epithelial growth factor receptor (EGFR) and found that compared to scFv-based CAR, T cells engineered with adnectin-based CARs exhibited equivalent cell-killing activity against target H292 lung cancer cells in vitro and had comparable antitumor efficacy in xenograft tumor-bearing mice in vivo. In addition, with optimal affinity tuning, adnectin-based CAR showed higher selectivity on target cells with high EGFR expression than on those with low expression. This new design of adnectin CARs can potentially facilitate the development of T cell immunotherapy for cancer and other diseases.