An IQ Consortium Perspective on Best Practices for Bioanalytical and Immunogenicity Assessment Aspects of CAR-T and TCR-T Cellular Therapies Development.

CAR-T therapies have shown remarkable efficacy against hematological malignancies in the clinic over the last decade and new studies indicate that progress is being made to use these novel therapies to target solid tumors as well as treat autoimmune disease. Innovation in the field, including TCR-T, allogeneic or "off the shelf" CAR-T, and autoantigen/armored CAR-Ts are likely to increase the efficacy and applications of these therapies. The unique aspects of these cell-based therapeutics; patient-derived cells, intracellular expression, in vivo expansion, and phenotypic changes provide unique bioanalytical challenges to develop pharmacokinetic and immunogenicity assessments. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) Translational and ADME Sciences Leadership Group (TALG) has brought together a group of industry experts to discuss and consider these challenges. In this white paper, we present the IQ consortium perspective on the best practices and considerations for bioanalytical and immunogenicity aspects toward the optimal development of CAR-T and TCR-T cell therapies.

Increased tongue use enhances 5-HT2C receptor immunostaining in hypoglossal motor nucleus.

Hypoglossal (XII) motoneurons are activated by type 2 receptors for serotonin (5-HT). This activation is especially strong during wakefulness which facilitates diverse motor functions of the tongue, including the maintenance of upper airway patency in obstructive sleep apnea (OSA) patients. We tested whether 5-HT2 receptor levels in the XII nucleus vary with intensity of tongue use. Three groups of rats were housed overnight under conditions of increasing oromotor activity: W-water available ad lib; S-sweetened water to stimulate drinking; S + O-sweetened water + oil applied on fur to increase grooming. After the exposures, immunostaining for 5-HT2C, but not 5-HT2A, receptors was higher in the XII nucleus in S + O than in W rats (65 ± 1.8 (SE) vs. 60 ± 2.0 arbitrary units; p = 0.008). In the medullary raphé obscurus region, the percentage of c-Fos-positive 5-HT cells was 13% higher (p = 0.03) in S + O than in W rats. The positive feedback between tongue use and 5-HT2C receptor immunostaining reveals a novel mechanism potentially relevant for OSA and neuromuscular disorders.

Reduced c-Fos expression in medullary catecholaminergic neurons in rats 20 h after exposure to chronic intermittent hypoxia.

Persons affected by obstructive sleep apnea (OSA) have increased arterial blood pressure and elevated activity in upper airway muscles. Many cardiorespiratory features of OSA have been reproduced in rodents subjected to chronic-intermittent hypoxia (CIH). We previously reported that, following exposure to CIH, rats have increased noradrenergic terminal density in brain stem sensory and motor nuclei and upregulated expression of the excitatory α(1)-adrenergic receptors in the hypoglossal motor nucleus. This suggested that CIH may enhance central catecholaminergic transmission. We now quantified c-Fos expression in different groups of pontomedullary catecholaminergic neurons as an indirect way of assessing their baseline activity in rats subjected to CIH or sham treatment (7 AM-5 PM daily for 35 days). One day after the last CIH exposure, the rats were gently kept awake for 2.5 h and then were anesthetized and perfused, and their pontomedullary brain sections were subjected to dopamine ß-hydroxylase (DBH) and c-Fos immunohistochemistry. DBH-positive cells were counted in the A1/C1, A2/C2, A5, subcoeruleus (sub-C) and A7 groups of catecholaminergic neurons, and the percentages of those expressing c-Fos were determined. We found that fewer DBH cells expressed c-Fos in CIH- than in sham-treated rats in the medulla (significant in the A1 group). In the pons (rostral A5, sub-C, and A7), c-Fos expression did not differ between the CIH- and sham-treated animals. We suggest that, when measured 20 h after the last CIH exposure, catecholaminergic transmission is enhanced through terminal sprouting and receptor upregulation rather than through increased baseline activity in pontomedullary catecholaminergic neurons.