Structure and function of a dual antagonist of the human growth hormone and prolactin receptors with site-specific PEG conjugates.

Human growth hormone (hGH) is a pituitary-derived endocrine protein that regulates several critical postnatal physiologic processes including growth, organ development, and metabolism. Following adulthood, GH is also a regulator of multiple pathologies like fibrosis, cancer, and diabetes. Therefore, there is a significant pharmaceutical interest in developing antagonists of hGH action. Currently, there is a single FDA-approved antagonist of the hGH receptor (hGHR) prescribed for treating patients with acromegaly and discovered in our laboratory almost 3 decades ago. Here, we present the first data on the structure and function of a new set of protein antagonists with the full range of hGH actions-dual antagonists of hGH binding to the GHR as well as that of hGH binding to the prolactin receptor. We describe the site-specific PEG conjugation, purification, and subsequent characterization using MALDI-TOF, size-exclusion chromatography, thermostability, and biochemical activity in terms of ELISA-based binding affinities with GHR and prolactin receptor. Moreover, these novel hGHR antagonists display distinct antagonism of GH-induced GHR intracellular signaling in vitro and marked reduction in hepatic insulin-like growth factor 1 output in vivo. Lastly, we observed potent anticancer biological efficacies of these novel hGHR antagonists against human cancer cell lines. In conclusion, we propose that these new GHR antagonists have potential for development towards multiple clinical applications related to GH-associated pathologies.

SON-1210 - a novel bifunctional IL-12 / IL-15 fusion protein that improves cytokine half-life, targets tumors, and enhances therapeutic efficacy.

Background: The potential synergy between interleukin-12 (IL-12) and IL-15 holds promise for more effective solid tumor immunotherapy. Nevertheless, previous clinical trials involving therapeutic cytokines have encountered obstacles such as short pharmacokinetics, limited tumor microenvironment (TME) targeting, and substantial systemic toxicity. Methods: To address these challenges, we fused single-chain human IL-12 and native human IL-15 in cis onto a fully human albumin binding (FHAB) domain single-chain antibody fragment (scFv). This novel fusion protein, IL12-FHAB-IL15 (SON-1210), is anticipated to amplify the therapeutic impact of interleukins and combination immunotherapies in human TME. The molecule was studied in vitro and in animal models to assess its pharmacokinetics, potency, functional characteristics, safety, immune response, and efficacy. Results: SON-1210 demonstrated robust binding affinity to albumin and exhibited the anticipated in vitro activity and tumor model efficacy that might be expected based on decades of research on native IL-12 and IL-15. Notably, in the B16F10 melanoma model (a non-immunogenic, relatively "cold" tumor), the murine counterpart of the construct, which had mouse (m) and human (h) cytokine sequences for the respective payloads (mIL12-FHAB-hIL15), outperformed equimolar doses of the co-administered native cytokines in a dose-dependent manner. A single dose caused a marked reduction in tumor growth that was concomitant with increased IFNγ levels; increased Th1, CTL, and activated NK cells; a shift in macrophages from the M2 to M1 phenotype; and a reduction in Treg cells. In addition, a repeat-dose non-human primate (NHP) toxicology study displayed excellent tolerability up to 62.5 µg/kg of SON-1210 administered three times, which was accompanied by the anticipated increases in IFNγ levels. Toxicokinetic analyses showed sustained serum levels of SON-1210, using a sandwich ELISA with anti-IL-15 for capture and biotinylated anti-IL-12 for detection, along with sustained IFNγ levels, indicating prolonged kinetics and biological activity. Conclusion: Collectively, these findings support the suitability of SON-1210 for patient trials in terms of activity, efficacy, and safety, offering a promising opportunity for solid tumor immunotherapy. Linking cytokine payloads to a fully human albumin binding domain provides an indirect opportunity to target the TME using potent cytokines in cis that can redirect the immune response and control tumor growth.

Site specific discrete PEGylation of (124)I-labeled mCC49 Fab' fragments improves tumor MicroPET/CT imaging in mice.

The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2-4 kDa discrete, branched PEGylation reagents on mCC49 Fab' (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab' (Fab'-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG 12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 ((124)I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab' with Mal-dPEG-A (Fab'-A) reduced the binding affinity of the non PEGylated Fab' by 30%; however, in vivo, Fab'-A significantly lengthened the blood retention vs Fab'-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab'-NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A.