Corrigendum to "Rational engineering of an improved adenosine deaminase 2 enzyme for weaponizing T-cell therapies": [Immuno-Oncology and Technology 10 (2023) 100394].

[This corrects the article DOI: 10.1016/j.iotech.2023.100394.].

Rational engineering of an improved adenosine deaminase 2 enzyme for weaponizing T-cell therapies.

Adenosine is a potent immunosuppressive metabolite that accumulates in the extracellular space within solid tumors and inhibits the antitumor function of native immune cell responses as well as chimeric antigen receptor (CAR) T-cell therapies. Here, we show that engineered human cells can degrade extracellular adenosine through secretion of adenosine deaminase (ADA) enzymes-a possible therapeutic enhancement for CAR T cells. We first determine that the high-activity ADA1 isoform is naturally intracellularly restricted and show that the addition of canonical or computationally predicted secretory peptides did not allow for improved secretion. We did, however, determine that the lower-activity ADA2 isoform is naturally secreted. Thus, we utilized phylogenetic-based structural comparisons to guide a mutational survey of ADA2 active site residues, which when coupled with a high-throughput screen for enhanced ADA2-mediated extracellular adenosine rate allowed isolation of the most catalytically efficient ADA2 variant reported to date. When expressed by human cells, this variant exhibits 30× higher extracellular adenosine degradation activity than the wild-type enzyme. Finally, we demonstrate that Jurkat and CAR T cells engineered to express this secreted, high-activity ADA2 variant can degrade significant amounts of extracellular adenosine in vitro.

Contemporary imaging of patients with a renal mass: does size on computed tomography equal pathological size?

OBJECTIVE: To evaluate the difference between radiographic size on computed tomography (CT) and the pathological size of renal tumours, in contemporary patients. PATIENTS AND METHODS: We retrospectively reviewed the records of 521 patients undergoing surgical resection of a renal mass between 2000 and 2007, who had tumour sizes recorded from both preoperative CT and pathological evaluation of the tumour specimen. Data on histological tumour type were also extracted. The paired Student's t-test was used to compare the mean radiographic size as measured on CT with the mean pathological size, with P < 0.05 considered to indicate statistical significance. RESULTS: For all patients, the mean radiographic size and mean pathological size was 4.79 and 4.69 cm, respectively (P = 0.02). Therefore, on average, radiographic size overestimated pathological size by 1 mm. In patients with a tumour of 4-7 cm, radiographic size overestimated pathological size by 0.21 cm (P = 0.007). However, there was no significant difference in patients with a tumour of <4 cm or >7 cm. CONCLUSIONS: Using contemporary patients, there was a statistically significant overestimation of renal tumour sizes by CT compared with the pathological assessment. However, the overall difference between radiographic and pathological tumour size was 1 mm, suggesting that CT provides an accurate method with which to estimate renal tumour size.

Cellular uptake and in situ binding of a peptide agonist for calmodulin.

We have used the method of inverted hydropathy to develop peptides that interact with EF hands of calmodulin (CaM). Previously we have shown these peptides specifically interact with their desired target in a productive manner, in that they activated CaM in the absence of Ca(2+). Therefore, we sought to determine whether these peptides would enter cells, remain intact, and interact with CaM in the interior of the cell. Using several techniques we have demonstrated cellular uptake, stability, and an intracellular interaction with CaM with fluorescein-labeled and radiolabeled peptides in Jurkat T cells. The results suggest that these peptides may be useful in the study and the manipulation of Ca(2+)-mediated pathways in cells.

De novo design of peptides targeted to the EF hands of calmodulin.

This report describes the use of the concept of inversion of hydropathy patterns to the de novo design of peptides targeted to a predetermined site on a protein. Eight- and 12-residue peptides were constructed with the EF hands or Ca(2+)-coordinating sites of calmodulin as their anticipated points of interaction. These peptides, but not unrelated peptides nor those with the same amino acid composition but a scrambled sequence, interacted with the two carboxyl-terminal Ca(2+)-binding sites of calmodulin as well as the EF hands of troponin C. The interactions resulted in a conformational change whereby the 8-mer peptide-calmodulin complex could activate phosphodiesterase in the absence of Ca(2+). In contrast, the 12-mer peptide-calmodulin complex did not activate phosphodiesterase but rather inhibited activation by Ca(2+). This inhibition could be overcome by high levels of Ca(2+). Thus, it would appear that the aforementioned concept can be used to make peptide agonists and antagonists that are targeted to predetermined sites on proteins such as calmodulin.