BackgroundFor their application in the area of diagnosis and therapy, single-domain antibodies (sdAbs) offer multiple advantages over conventional antibodies and fragments thereof in terms of size, stability, solubility, immunogenicity, production costs as well as tumor uptake and blood clearance. Thus, sdAbs have been identified as valuable next-generation targeting moieties for molecular imaging and drug delivery in the past years. Since these probes are much less complex than conventional antibody fragments, bacterial expression represents a facile method in order to produce sdAbs in large amounts as soluble and functional proteins.ResultsBy the combined use of high cell density cultivation media with a genetically engineered E. coli mutant strain designed for the cytoplasmic formation of proper disulfide bonds, we achieved high level of intracellular sdAb production (up to 200 mg/L). Due to a carboxyterminal hexahistidine epitope, the soluble recombinant sdAbs could be purified by one-step immobilized metal affinity chromatography to apparent homogeneity and easily radiolabeled with 99mTc within 1 h. The intradomain disulfide bridge being critical for the stability and functionality of the sdAb molecule was shown to be properly formed in ~96% of the purified proteins. In vitro binding studies confirmed the high affinity and specificity of the expressed sdAb 7C12 towards its molecular target.ConclusionsOur study demonstrates an efficient cultivation and expression strategy for the production of substantial amounts of soluble and functional sdAbs, which may be adopted for high-yield production of other more complex proteins with multiple disulfides as well.

The approval of tyrosine kinase inhibitors and checkpoint inhibitors represented a remarkable progression in the therapeutic landscape for the treatment of metastatic renal cell carcinoma (mRCC). Yet, in the ever-evolving landscape of mRCC treatment, real-world data on these agents, including pazopanib, are scarce. The non-interventional PAZOREAL study investigated the effectiveness and safety of pazopanib (first-line), nivolumab (second-line), and everolimus (second- and third-line) in a real-life setting. The multicentric study included 376 mRCC patients who received first-line treatment with pazopanib and assessed time on the drug (primary endpoint), overall survival, best responses, disease control rates, as well as safety signals and health-related quality of life. The median overall time on the drug was 10.0 months, with first-line pazopanib having a median time on drug of 6.3 months. The median overall survival was 35.9 months. The disease control rate for first-line pazopanib was 56.9%. No new safety signals were detected. PAZOREAL provides valuable real-world data for first-line treatment with pazopanib.

e16511 Background: In a real-world setting, a substantial number of patients (pts) with metastatic renal cell carcinoma (mRCC) do not meet all eligibility criteria for a clinical trial. Thus, data on effectiveness and safety of 1st-line (1L) treatment with pazopanib (PAZO) as well as quality of life (QoL) is underrepresented for those pts and data on treatment sequences implementing PAZO as 1L are sparse. Methods: PAZOREAL is a prospective, multi-center, non-interventional study to evaluate effectiveness, safety and QoL in pts with mRCC treated with 1L PAZO, and nivolumab (NIVO) or everolimus in second and third line. In this subgroup analysis, the clinical outcome of trial-eligible (TE) and trial-ineligible (TIE) pts with mRCC was assessed by means of overall survival (OS), time on drug (ToD), treatment-emergent adverse events (TEAE) and QoL evaluated by EQ-5D-5L. Pts were rated TIE if they met at least one of the following three ‘TIE criteria’: (i) Karnofsky Performance Status < 70%, (ii) hemoglobin below the lower limit of normal, and (iii) non-clear cell carcinoma1. Results: Of 398 pts treated between December 2015 and February 2021, 376 pts received 1L treatment with PAZO. The median age was 69.7 years. 146 pts were categorized TE and 184 pts TIE, for 46 pts assessment was not applicable. Most of all pts were initially treated with PAZO 800 mg (TE: 71.9% vs TIE: 63.0%). Median ToD for PAZO was 7.7 months (95% CI 6.1-9.0) for TE and 6.0 months (95% CI 4.5-8.1) for TIE. A similar fraction of TE and TIE pts were treated with 2L NIVO (43.8% vs 44.0%). Median OS was 53.2 months (95% CI 38.9-NA) for TE and 26.0 months (95% CI 17.3-35.9) for TIE. The 12-month OS rate was 77.9% for TE and 67.1% for TIE. In patient reported outcome (PRO) measures, baseline QoL was estimated higher for TE than TIE, i.e., 63.2% TE vs 49.2% TIE pts had no limitations for mobility and 82.9% TE vs 61.7% TIE pts were independent in self-care. Frequency of related TEAEs grade 3/4 were comparable for TE and TIE (26.7% vs 25.0%), while TE tended to have less related serious TEAEs (13.0% vs 16.3%). Treatment was discontinued due to related TEAEs in 19.2% of TE and 13.0% of TIE pts. Conclusions: PAZOREAL provides real-world data for mRCC pts usually not represented in clinical trials during the study period. As it was to be expected, median OS and PRO were more favorable for TE than for the TIE pts, reflecting the difference in general condition between these groups. But more importantly, these data underline the tolerability of 1L PAZO treatment even for TIE pts, which is supported by comparable ToD for 1L PAZO and the comparable safety profile in both subgroups.1 Marschner, N. et al. Clin Genitourin Cancer 2017; 15(2):e209-215. doi:10.1016/j.clgc.2016.08.022.