The lymphocytes of epithelial and lamina proprial compartments of the intestine are phenotypically and functionally distinct and serve a wide range of functions in the intestinal mucosa like regulating intestinal homeostasis, maintaining epithelial barrier function as well as regulating adaptive and innate immune responses. To analyze the role of these cells in different disease states, it is necessary to isolate pure cell populations of the intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) of the gut. In this protocol we describe a method to isolate T cells from IEL and LPL, which can be used for further investigations like comparative studies of mRNA expression, cell proliferation assay, or protein analysis.

With the routine use of effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, the number of life-threatening coronavirus disease 2019 (COVID-19) courses have largely been reduced. However, multiple COVID-19 convalescents, even after asymptomatic to moderate disease, suffer from post-COVID syndrome, with relevant limitations in daily life. The pathophysiologic mechanisms of post-COVID syndrome are still elusive, with dysregulation of the immune system suggested as a central mechanism. Here, we assessed COVID-19 post-infectious symptoms (5–6 months after PCR-confirmed acute infection) together with the humoral immune response against SARS-CoV-2 in non-hospitalized COVID-19 convalescents, early (5–6 weeks) and late (5–6 months) after their first positive SARS-CoV-2 PCR result. Convalescents reporting several post-infectious symptoms (>3) showed higher anti-spike and anti-nucleocapsid antibody levels 5–6 weeks after PCR-confirmed infection with the latter remained increased 5–6 months after positive PCR. Likewise, a higher post-infectious symptom score was associated with increased antibody levels. Of note, convalescents displaying neuro-psychiatric symptoms such as restlessness, palpitations, irritability, and headache, as well as general symptoms such as fatigue/reduced power had higher SARS-CoV-2-specific antibody levels compared with asymptomatic cases. The increased humoral immune response in convalescents with post-COVID syndrome might be useful for the detection of individuals with an increased risk for post-COVID syndrome.

TPS5114 Background: Bispecific antibodies (bsAbs), alike CAR T cells, are not yet successful in solid tumors. Recently we developed a PSMAxCD3 bsAb termed CC-1 in an IgG-based format mediating fully target cell-restricted T cell activation and potent antitumor activity (Zekri et al, EMBO Mol Med, 2020). In prostate cancer (PC), PSMA is not only expressed on the malignant cells themselves, but also on tumor vessels. Targeting the latter improves accessibility of the tumor site for immune effector cells, a critical prerequisite for success. CC-1 is under evaluation in a First in Human trial enrolling patients with castration resistant prostate carcinoma (NCT04104607) and, in combination with checkpoint inhibition, in a phase I trial enrolling patients with squamous cell carcinoma of the lung (NCT04104607). On the basis of the meanwhile available very favorable safety and preliminary efficacy data, CC-1 is now being evaluated as first line treatment in patients with biochemical recurrence (BCR) of PC (NCT05646550), where tumor burden is low and accordingly further reduced side effects and sustained efficacy are expected. Methods: This is an ongoing open label, single center phase I clinical trial evaluating CC-1 without androgen deprivation therapy as first line treatment in men with BCR of PC. Key eligibility criteria include low risk of disease progression (PSA-Doubling time > 1 year after prostatectomy or interval to BCR > 18 months after radiation; ISUP grade < 4; PSA ≥ 0.2 ng/ml and < 5 ng/ml) and no androgen deprivation therapy except in a neoadjuvant/adjuvant setting. The clinical trial consists of (i) a dose escalation part (24-42 patients) using a 3+3 design to determine overall safety, tolerability as well as maximum tolerated dose (MTD) and (ii) a dose expansion part, in which 14 patients are treated at the MTD. CC-1 is applied twice-weekly over 21 days as three-hour infusion in up to six 28-day cycles. In the first cycle, step dosing (day 1: 10µg, day 2: 28µg, day 3 and thereafter: target dose) is performed, and 7 patient cohorts receiving dose levels from 78µg to 600µg are evaluated. Premedication includes acetaminophen, dimetindene and cortisone to minimize the risk of infusion reactions and cytokine release syndrome (CRS). The primary objectives are definition of MTD and recommended phase II dose. Assessment of safety is based on the frequency of adverse events according to CTCAE v5.0. Dose limiting toxicities are defined as ≥ 3° adverse drug reactions (e.g. CRS) with predefined exceptions (e.g. temporary laboratory abnormalities). Efficacy is determined by serial PSA-measurements (PSA-response: ≥50% decrease compared to baseline). Furthermore, percentage of patients with no clinical relapse, no salvage and no subsequent antineoplastic therapy will be assessed. Clinical trial information: NCT05646550 .

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2534 Background: While being highly efficient in hematological malignancies, bispecific antibodies (bsAbs), alike CAR T cells, are yet not successful in solid tumors. Moreover, all available T cell-mobilizing strategies cause side effects that endanger patients and, in case of bsAbs, limit applicable doses and thus efficacy. We report on the clinical development of CC-1, a PSMAxCD3 bsAb, in an IgG-based format that induces fully target cell-restricted T cell activity (Zekri et al, EMBO Mol Med, 2020). Targeting PSMA, which is expressed on malignant cells and on the neovasculature, improves accessibility of the tumor site for immune effector cells as critical prerequisite for success in solid tumors. Notably, CC-1 binds a unique PSMA epitope which is expressed on malignant cells both of prostate carcinoma (PC) and 50% of patients with squamous cell carcinoma (SCC) of the lung. Methods: A FIH trial evaluating CC-1 with pre-emptive Tocilizumab included patients with metastatic castration resistant prostate carcinoma (mCRPC) (NCT04104607) and consisted of two parts. Dose escalation (n=10-66) using a novel intra-individual dose escalation design to rapidly reach the target dose of 826µg to determine safety, tolerability and maximum tolerated dose (MTD) (Labrenz et al, Pharm Stat, 2022). The dose expansion cohort exposed patients to CC-1 at MTD and explored efficacy to define RP2D (n=14). Our second phase I trial enrolls patients with SCC of the lung (NCT04496674) to receive CC-1 in combination with checkpoint inhibition. Based on the meanwhile available very favorable safety and preliminary efficacy data, a third phase I trial was initiated where CC-1 is evaluated as first line treatment in patients with hormone sensitive biochemical recurrence (BCR) of PC (NCT05646550), where tumor burden is low and accordingly lower side effects and long-lasting efficacy are expected. Results: Recruitment in the dose escalation part of the trial in mCRPC has been completed and the target dose was reached and MTD defined without DLT upon treatment of the 9th and 14th patient, respectively. 24 patients completed treatment, with the most frequently observed toxicity being cytokine release syndrome (CRS, max. 2°) (88%). Besides grade 1 to 2 hypertension (46%) and xerostomia (8%), no further CC-1 related toxicities were observed. A rapid and profound decline of elevated PSA levels was observed in all but one patient, with up to 60% reduction compared to baseline. So far 5 patients received multiple treatment cycles at MTD-level. One patient with SCC of the lung has been treated and CC-1 was escalated up to 153µg without occurrence of CRS. In the phase I trial in BCR of PC, the first three patients were enrolled in February 2023. Conclusions: CC-1 is a promising compound with a favourable toxicity profile and promising clinical activity. Details on study designs and updated data from the 3 clinical trials will be presented at the meeting. Clinical trial information: NCT04104607 , NCT04496674 , NCT05646550 .