Targeting the ion channel Kv1.3 with scorpion venom peptides engineered for potency, selectivity, and half-life.

Ion channels are an attractive class of drug targets, but progress in developing inhibitors for therapeutic use has been limited largely due to challenges in identifying subtype selective small molecules. Animal venoms provide an alternative source of ion channel modulators, and the venoms of several species, such as scorpions, spiders and snails, are known to be rich sources of ion channel modulating peptides. Importantly, these peptides often bind to hyper-variable extracellular loops, creating the potential for subtype selectivity rarely achieved with small molecules. We have engineered scorpion venom peptides and incorporated them in fusion proteins to generate highly potent and selective Kv1.3 inhibitors with long in vivo half-lives. Kv1.3 has been reported to play a role in human T cell activation, and therefore, these Kv1.3 inhibitor fusion proteins may have potential for the treatment of autoimmune diseases. Our results support an emerging approach to generating subtype selective therapeutic ion channel inhibitors.

Structural insights into the unique pH-responsive characteristics of the anti-TIGIT therapeutic antibody Ociperlimab.

TIGIT is mainly expressed on T cells and is an inhibitory checkpoint receptor that binds to its ligand PVR in the tumor microenvironment. Anti-TIGIT monoclonal antibodies (mAbs) such as Ociperlimab and Tiragolumab block the TIGIT-PVR interaction and are in clinical development. However, the molecular blockade mechanism of these mAbs remains elusive. Here, we report the crystal structures of TIGIT in complex with Ociperlimab_Fab and Tiragolumab_Fab revealing that both mAbs bind TIGIT with a large steric clash with PVR. Furthermore, several critical epitopic residues are identified. Interestingly, the binding affinity of Ociperlimab toward TIGIT increases approximately 17-fold when lowering the pH from 7.4 to 6.0. Our structure shows a strong electrostatic interaction between ASP103HCDR3 and HIS76TIGIT explaining the pH-responsive mechanism of Ociperlimab. In contrast, Tiragolumab does not show an acidic pH-dependent binding enhancement. Our results provide valuable information that could help to improve the efficacy of therapeutic antibodies for cancer treatment.

Identification and characterization of an unexpected isomerization motif in CDRH2 that affects antibody activity.

Aspartic acid (Asp) isomerization is a spontaneous non-enzymatic post-translation modification causing a change in the structure of the protein backbone, which is commonly observed in therapeutic antibodies during manufacturing and storage. The Asps in Asp-Gly (DG), Asp-Ser (DS), and Asp-Thr (DT) motifs in the structurally flexible regions, such as complementarity-determining regions (CDRs) in antibodies, are often found to have high rate of isomerization, and they are considered "hot spots" in antibodies. In contrast, the Asp-His (DH) motif is usually considered a silent spot with low isomerization propensity. However, in monoclonal antibody mAb-a, the isomerization rate of an Asp residue, Asp55, in the aspartic acid-histidine-lysine (DHK) motif present in CDRH2 was found to be unexpectedly high. By determining the conformation of DHK motif in the crystal structure of mAb-a, we found that the Cgamma of the Asp side chain carbonyl group and the back bone amide nitrogen of successor His were in proximal contact, which facilitates the formation of succinimide intermediate, and the +2 Lys played an important role in stabilizing such conformation. The contributing roles of the His and Lys residues in DHK motif were also verified using a series of synthetic peptides. This study identified a novel Asp isomerization hot spot, DHK, and the structural-based molecular mechanism was revealed. When 20% Asp55 isomerization in this DHK motif occurred in mAb-a, antigen binding activity reduced to 54%, but the pharmacokinetics in rat was not affected significantly. Although Asp isomerization of DHK motif in CDR does not appear to have a negative impact on PK, DHK motifs in the CDRs of antibody therapeutics should be removed, considering the high propensity of isomerization and impact on antibody activity and stability.

Accelerating the speed of innovative anti-tumor drugs to first-in-human trials incorporating key de-risk strategies.

Pharmaceutical companies have recently focused on accelerating the timeline for initiating first-in-human (FIH) trials to allow quick assessment of biologic drugs. For example, a stable cell pool can be used to produce materials for the toxicology (Tox) study, reducing time to the clinic by 4-5 months. During the coronavirus disease 2019 (COVID-19) pandemic, the anti-COVID drugs timeline from DNA transfection to the clinical stage was decreased to 6 months using a stable pool to generate a clinical drug substrate (DS) with limited stability, virus clearance, and Tox study package. However, a lean chemistry, manufacturing, and controls (CMC) package raises safety and comparability risks and may leave extra work in the late-stage development and commercialization phase. In addition, whether these accelerated COVID-19 drug development strategies can be applied to non-COVID projects and established as a standard practice in biologics development is uncertain. Here, we present a case study of a novel anti-tumor drug in which application of "fast-to-FIH" approaches in combination with BeiGene's de-risk strategy achieved successful delivery of a complete CMC package within 10 months. A comprehensive comparability study demonstrated that the DS generated from a stable pool and a single-cell-derived master cell bank were highly comparable with regards to process performance, product quality, and potency. This accomplishment can be a blueprint for non-COVID drug programs that approach the pace of drug development during the pandemic, with no adverse impact on the safety, quality, and late-stage development of biologics.

"Stapling" scFv for multispecific biotherapeutics of superior properties.

Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation ("breathing") and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.

GFRAL is the receptor for GDF15 and the ligand promotes weight loss in mice and nonhuman primates.

Growth differentiation factor 15 (GDF15), a distant member of the transforming growth factor (TGF)-ß family, is a secreted protein that circulates as a 25-kDa dimer. In humans, elevated GDF15 correlates with weight loss, and the administration of GDF15 to mice with obesity reduces body weight, at least in part, by decreasing food intake. The mechanisms through which GDF15 reduces body weight remain poorly understood, because the cognate receptor for GDF15 is unknown. Here we show that recombinant GDF15 induces weight loss in mice fed a high-fat diet and in nonhuman primates with spontaneous obesity. Furthermore, we find that GDF15 binds with high affinity to GDNF family receptor α-like (GFRAL), a distant relative of receptors for a distinct class of the TGF-ß superfamily ligands. Gfral is expressed in neurons of the area postrema and nucleus of the solitary tract in mice and humans, and genetic deletion of the receptor abrogates the ability of GDF15 to decrease food intake and body weight in mice. In addition, diet-induced obesity and insulin resistance are exacerbated in GFRAL-deficient mice, suggesting a homeostatic role for this receptor in metabolism. Finally, we demonstrate that GDF15-induced cell signaling requires the interaction of GFRAL with the coreceptor RET. Our data identify GFRAL as a new regulator of body weight and as the bona fide receptor mediating the metabolic effects of GDF15, enabling a more comprehensive assessment of GDF15 as a potential pharmacotherapy for the treatment of obesity.

Engineering peptide therapeutics using MIMETIBODY™ technology.

The MIMETIBODY™ platform was developed to expand the opportunities for application of biotherapeutics. While the utility of antibodies as antagonists has been well demonstrated, their application as agonists has been more challenging. For steric reasons, antibodies may be less well suited to perform as agonists or as inhibitors of GPCRs. In contrast, many bioactive peptides function as agonists or by interaction with GPCRs but their development as therapeutics has been challenging due to their small size and metabolic lability. The MIMETIBODY™ platform has been used to develop a variety of stable, long-lived molecules with intrinsic activities similar to that of their parent peptides. This chapter describes methods for construction of expression plasmids, expression and purification strategies, and methods for characterizing the activity of these novel proteins.

CNTO 530 increases expression of HbA and HbF in murine models of ß-thalassemia and sickle cell anemia.

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of ß-thalassemia and sickle cell anemia (Berkeley mice). ß- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human ß-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in ß- thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of ß-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies.

Receptor-Fc fusion therapeutics, traps, and MIMETIBODY technology.

Fc fusion proteins are molecules in which the immunoglobulin Fc is fused genetically to a protein of interest, such as an extracellular domain of a receptor, ligand, enzyme, or peptide. Fc fusion proteins have some antibody-like properties such as long serum half-life and easy expression and purification, making them an attractive platform for therapeutic drugs. Five Fc fusion based drugs are on the market presently, and many more are in different stages of clinical trials, demonstrating that Fc fusion proteins have become credible alternatives to monoclonal antibodies as therapeutics. This review summarizes the Fc fusion proteins that have been approved for use in the clinic and those that are currently in clinical trials, as well as the different approaches to design Fc fusion proteins.