Vendor and in-house verification identified two variants in the scFv CDS: a 1-bp deletion of G at nt 755 and a C→T transition at nt 1163.By aligning Sanger reads to the codon-optimized reference using NCBI BLASTn, we confirmed both events. Results revealed a 1-bp G deletion at nt755, producing a frameshift and predicted premature stop, which compromises downstream domains and likely disrupts the C-terminal 6×His tag. We also observed a C→T substitution at nt1163; its protein-level consequence depends on codon context (synonymous vs. missense vs. nonsense) but does not alter the decision once the frameshift is present. Due to the observed deletion and mutation (1-bp deletion causing a frameshift), we will not proceed with functional assays for this construct.
To support these findings, we provide BLAST alignment snapshots and Sanger chromatograms:
BLAST
Alignment snapshot around the predefined coordinate (nt755) with Query = Sanger read and Subject = codon-optimized reference CDS. The dash (–) indicates a single-base gap relative to the reference, consistent with a 1-bp deletion in the insert that would shift the reading frame and predict a downstream premature stop.
Alignment snapshot around the predefined coordinate (nt1163) with Query = Sanger read and Subject = codon-optimized reference CDS. A single base differs between the two sequences (C→T in the read), consistent with the reported SNP at nt1163.
Sanger
To confirm that our constructs were successfully expressed in mammalian cells, we transfected HEK293T cells with five different anti-IL-23 scFv prototypes. Among them, three were originally designed antibodies, generated by assembling sequences identified through extensive database research, while the other two were scFv versions of clinically used antibody drugs. After 48h, cell lysates were analyzed by Western blot using an anti-His antibody. We observed clear bands at ~25 and ~50 kDa, corresponding to the expected molecular weight of the scFv. These results confirmed that our constructs were expressed correctly in HEK293T cells.
To evaluate whether our constructs were secreted into the extracellular medium, we analyzed both the supernatants from our own HEK293T transfection and the purified proteins expressed by an outsourced HEK293 expression system.
In our lab-scale HEK293T transfection experiments, supernatants were collected after transfection and concentrated using Amicon Ultra-0.5 centrifugal filters (3 kDa cutoff) to enhance detection. Western blot analysis revealed that only the two scFv constructs derived from clinically used antibody drugs were detected in the supernatant, while the three originally designed prototypes showed no visible secretion signals. Interestingly, Western blot analysis of the cell lysates revealed both full-length and cleaved forms of the prototype scFv, suggesting that the signal peptide might have been prematurely removed, preventing proper secretion.
However, when the same constructs were expressed and purified by a professional production service using the HEK293 expression system and Ni-NTA affinity chromatography, all three designed scFv variants were successfully obtained from the culture supernatant. This result suggests that the lack of secretion in our HEK293T setup was primarily due to expression system limitations — such as the lower secretion efficiency of HEK293T cells, and premature signal peptide cleavage, rather than intrinsic sequence defects.
Together, these findings indicate that all five scFv constructs are in principle capable of secretion under optimized expression conditions, and that differences in processing efficiency between HEK293T and HEK293 systems may critically affect successful protein export.
To assess whether our scFv constructs retained their ability to recognize IL-23, we conducted a series of ELISA experiments using three different sample types: (1) concentrated culture supernatants, (2) cell lysates, and (3) purified scFv proteins expressed by a commercial HEK293 system. In each assay, wells were coated with 5 ng of recombinant IL-23 per well as the capture antigen, following the protocol described in the Experiments – ELISA section.
Supernatant-based ELISA
Using concentrated supernatants from our in-house HEK293T transfection, ELISA assays showed that only the two scFv (p19, p40) constructs derived from clinically used antibody drugs displayed strong binding to IL-23 across two independent batches. In contrast, the three originally designed prototypes showed detectable signals only at the highest concentrations, indicating that these constructs may have low binding affinity to IL-23 or limited protein stability and expression efficiency.
Lysate-based ELISA
We next examined whether the intracellular scFv retained binding activity by performing ELISA with cell lysates. Due to protein loss caused by freeze–thaw treatment, only the first batch yielded usable samples. The results again showed binding exclusively for the drug-derived scFv constructs, consistent with the supernatant-based findings.
Purified scFv from commercial HEK293 expression
To further confirm the binding capability of our original scFv prototypes, we tested purified scFv1, scFv2, and scFv3 obtained from a professional HEK293 expression service using Ni-NTA affinity chromatography. At the highest tested concentration, scFv1 and scFv3 exhibited clear IL-23 binding signals, while scFv2 did not show a significant difference, likely due to its relatively lower concentration in the purified sample. These results demonstrate that at least two of our prototype scFv constructs can specifically recognize IL-23 once correctly expressed and processed.
Together, these findings indicate that while our in-house HEK293T system produced detectable binding only for the drug-derived constructs, the purified scFv expressed by the HEK293 system confirmed that scFv1 and scFv3 from our original designs also retain IL-23-binding capability, validating our design concept.
To determine the optimal conditions for downstream signaling analysis, we first examined the time-dependent activation of STAT3 in Caco-2 cells following IL-23 stimulation. Cells were treated with 20 ng/mL IL-23 for 15, 30, 60, and 180 minutes, and phosphorylation of STAT3 was analyzed by Western blot. p-STAT3 and p-JAK2 levels both increased at 30 minutes. Based on this result, the 30-minute timepoint was selected for subsequent co-treatment experiments.
To evaluate the functional effects of anti-IL-23 scFvs on downstream inflammatory signaling, we performed Western blot analysis on Caco-2 cells stimulated with IL-23 and co-treated with individual scFvs for 30 minutes. IL-23 stimulation alone led to a clear increase in p-STAT3 levels, confirming effective activation of the STAT3 pathway.
Among the tested scFvs, only p19 showed a partial inhibitory effect—p-STAT3 expression was reduced to near-baseline levels (1.02 relative to control). scFv #1 produced minimal suppression, with p-STAT3 levels similar to the IL-23–only group, indicating weak or insufficient activity. In contrast, p40 failed to inhibit signaling, raising the possibility of a crosslinking effect or unintended stabilization of the IL-23 receptor complex. Taken together, these results suggest that only p19 exhibits a measurable inhibitory effect on IL-23–induced inflammatory signaling, while the other scFvs either lack functional activity or may even enhance pathway activation.
