Enhanced therapeutic potential of antibody fragment via IEDDA-mediated site-specific albumin conjugation.

BACKGROUND: The use of single-chain variable fragments (scFvs) for treating human diseases, such as cancer and immune system disorders, has attracted significant attention. However, a critical drawback of scFv is its extremely short serum half-life, which limits its therapeutic potential. Thus, there is a critical need to prolong the serum half-life of the scFv for clinical applications. One promising serum half-life extender for therapeutic proteins is human serum albumin (HSA), which is the most abundant protein in human serum, known to have an exceptionally long serum half-life. However, conjugating a macromolecular half-life extender to a small protein, such as scFv, often results in a significant loss of its critical properties. RESULTS: In this study, we conjugated the HSA to a permissive site of scFv to improve pharmacokinetic profiles. To ensure minimal damage to the antigen-binding capacity of scFv upon HSA conjugation, we employed a site-specific conjugation approach using a heterobifunctional crosslinker that facilitates thiol-maleimide reaction and inverse electron-demand Diels-Alder reaction (IEDDA). As a model protein, we selected 4D5scFv, derived from trastuzumab, a therapeutic antibody used in human epithermal growth factor 2 (HER2)-positive breast cancer treatment. We introduced a phenylalanine analog containing a very reactive tetrazine group (frTet) at conjugation site candidates predicted by computational methods. Using the linker TCO-PEG4-MAL, a single HSA molecule was site-specifically conjugated to the 4D5scFv (4D5scFv-HSA). The 4D5scFv-HSA conjugate exhibited HER2 binding affinity comparable to that of unmodified 4D5scFv. Furthermore, in pharmacokinetic profile in mice, the serum half-life of 4D5scFv-HSA was approximately 12 h, which is 85 times longer than that of 4D5scFv. CONCLUSIONS: The antigen binding results and pharmacokinetic profile of 4D5scFv-HSA demonstrate that the site-specifically albumin-conjugated scFv retained its binding affinity with a prolonged serum half-life. In conclusion, we developed an effective strategy to prepare site-specifically albumin-conjugated 4D5scFv, which can have versatile clinical applications with improved efficacy.

Enhanced anti-tumor activity of arginine decarboxylase through the incorporation of aromatic amino acids at the multimer-forming interface.

The pressing challenge of cancer's high mortality and invasiveness demands improved therapeutic approaches. Targeting the nutrient dependencies within cancer cells has emerged as a promising approach. This study is dedicated to demonstrating the potential of arginine depletion for cancer treatment. Notably, the focus centers on arginine decarboxylase (RDC), a pH-dependent enzyme expecting enhanced activity within the slightly acidic microenvironments of tumors. To investigate the effect of a single-site mutation on the catalytic efficacy of RDC, diverse amino acids, including glycine, alanine, phenylalanine, tyrosine, tryptophan, p-azido-phenylalanine, and a phenylalanine analog with a hydrogen-substituted tetrazine, were introduced at the crucial threonine site (position 39) in the multimer-forming interface. Remarkably, the introduction of either a natural or a non-natural aromatic amino acid at position 39 substantially boosted enzymatic activity, while amino acids with smaller side chains did not show the same effect. This enhanced enzymatic activity is likely attributed to the reinforced formation of multimer structures through favorable interactions between the introduced aromatic amino acid and the neighboring subunit. Noteworthy, at slightly acidic pH, the RDC variant featuring tryptophan at position 39 demonstrated augmented cytotoxicity against tumor cells compared to the wild-type RDC. This attribute aligns with the tumor microenvironment and positions these variants as potential candidates for targeted cancer therapy.

Computation-Aided Design of Albumin Affibody-Inserted Antibody Fragment for the Prolonged Serum Half-Life.

Single-chain variable fragments (scFvs) have been recognized as promising agents in cancer therapy. However, short serum half-life of scFvs often limits clinical application. Fusion to albumin affibody (ABD) is an effective and convenient half-life extension strategy. Although one terminus of scFv is available for fusion of ABD, it is also frequently used for fusion of useful moieties such as small functional proteins, cytokines, or antibodies. Herein, we investigated the internal linker region for ABD fusion instead of terminal region, which was rarely explored before. We constructed two internally ABD-inserted anti-HER2 4D5scFv (4D5-ABD) variants, which have short (4D5-S-ABD) and long (4D5-L-ABD) linker length respectively. The model structures of these 4D5scFv and 4D5-ABD variants predicted using the deep learning-based protein structure prediction program (AlphaFold2) revealed high similarity to either the original 4D5scFv or the ABD structure, implying that the functionality would be retained. Designed 4D5-ABD variants were expressed in the bacterial expression system and characterized. Both 4D5-ABD variants showed anti-HER2 binding affinity comparable with 4D5scFv. Binding affinity of both 4D5-ABD variants against albumin was also comparable. In a pharmacokinetic study in mice, the 4D5-ABD variants showed a significantly prolonged half-life of 34 h, 114 times longer than that of 4D5scFv. In conclusion, we have developed a versatile scFv platform with enhanced pharmacokinetic profiles with an aid of deep learning-based structure prediction.

A case of metastatic squamous cell carcinoma arising from actinic cheilitis.

Actinic keratosis (AK) is a common, sun-induced, pre-malignant lesion with a strong likelihood of progressing to a malignancy. The reported risk of AK progressing to squamous cell carcinoma (SCC) varies from less than 1% to 20%. Clinically, induration, pain, large size, marked hyperkeratosis, ulceration, bleeding, rapid growth, and recurrence or persistence may be markers of AK progression into SCC. The risk of SCC metastasizing ranges between 0.5% and 3%. However, SCC of the lip arising from actinic cheilitis is more prone to metastasis than cutaneous SCC, with rates of the former varying between 3% and 20%. Here we report a typical case of SCC from actinic cheilitis with metastasis to the lymph nodes during a 4-year follow-up period. To exclude SCC, we emphasize the need for regular follow-up and prompt evaluation, including careful pathologic examination for actinic cheilitis.

A case of post-injury angiokeratoma circumscriptum of the chest.

Angiokeratomas represent vascular lesions and are histologically characterized by superficial vascular ectasia and overlying acanthosis and/or hyperkeratosis. Angiokeratomas can be classified into five types, with angiokeratoma circumscriptum representing the least common of the five types. Angiokeratoma circumscriptum presents at birth and frequently occurs unilaterally on the leg. Herein, we report a rare case of long term angiokeratoma circumscriptum (twelve years) on the right chest, discovered following the excision of a lipoma from the same area.

Formononetin promotes early fracture healing through stimulating angiogenesis by up-regulating VEGFR-2/Flk-1 in a rat fracture model.

Plant-derived phytoestrogens have bone protective effects, but the molecular mechanism behind these effects remains unclear. This study is aimed at fully characterizing the fracture healing process of formononetin, and investigating the mechanism underlying angiogenesis in calluses of a rat fracture model. Femoral fractures were produced in 2-month-old Sprague-Dawley rats. A 20 microg/kg or 200 microg/kg dose of formononetin was orally administrated once a day during the healing period of 21 days. The results showed that in the early stage of chondrogenesis (days 3), formononetin significantly increased the number of vessels, and expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2/flk-1) compared with control. However, the larger dose of formononetin had no significant difference on expression of VEGF and VEGFR-2/Flk-1 compared with that of the smaller dose of formononetin. After 7 days of administration, formononetin markedly induced differentiation of mesenchymal stem cells in the fracture site. After 14 days, gene expression of mesenchymal progenitors such as alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN) and collagen type I (Col I), indicating osteogenic differentiation, was markedly stimulated by formononetin compared with control. These results suggest that formononetin promotes early fracture healing through angiogenesis activation in the early stage of fracture repair, and osteogenesis acceleration in the later stages, and thus may be beneficial for fracture healing.