Abstract Background: Nanobodies have emerged as a versatile tool in imaging and therapy as a carrier of small molecules and radionuclides. Because of their small size, they act like "small molecules" in terms of rapid renal clearance but offer the high specificity of antibodies. However, similar to small molecule-based Targeted Radiation Therapy, nanobodies are reabsorbed/retained in the Kidney proximal tubules, leading to kidney toxicity (radiation nephropathy) by damaging renal cells. This dose-limiting toxicity may limit the maximum dose of radiation that is necessary to ablate tumors, leading to reduced effectiveness, failure to achieve complete tumor killing, and treatment resistance at a later stage. To overcome the limitation of the use of nanobodies as radionuclide carrier, we used a previously developed mesothelin-specific nanobody (JZQ-B4 against MSLN, Mesothelin) and modified its pharmacokinetics and linker chemistry, and demonstrated its potential as a carrier of a radioactive payload to a subcutaneous tumor (KPCY7160c2). Methods: B4 nanobody and its engineered variants were produced from E. Coli soluble fraction and purified by utilizing 6xHis-Tag/NiNTA followed by ion exchange chromatography. The molecules were labeled with Azide (N3) at a 1: 3 (protein: linker) ratio, then clicked with DFO-DBCO or DFO-Linker-DBCO (for Zr89 PET-imaging) ; or DOTA-DBCO or DOTA-Linker-DBCO (for Lu177/Ac225; β/α-therapy). To achieve our therapeutic goal, we adopted two molecular design strategies. First, we optimized the pharmacokinetics of B4 by fusing it to albuminbinding nanobodies engineered with varying affinities for serum albumin to achieve an optimal circulation halflife. Second, we systematically evaluated aminoacid linker architectures between the chelator (e. g. , DOTA, DFO) and DBCO to identify linker chemistries that enhance renal clearance of the radiolabeled constructs. Results: We observed that unmodified nanobody conjugated with radioisotope (B4-DFO (Zr89) ) bound tumors specific to mesothelin expression but was also taken up by the kidneys at high levels. This resulted in B4 uptake of 1% ID/g (Injected Dose/Gram) at the tumor, and as high as 20 % ID/g at the kidneys. The low uptake by the tumor is attributed to rapid clearance of nanobody in circulation (half-life less than 1 hr). With the fusion to high affinity serum albumin binder, the circulation half-life increased to 24 hours, resulting in accumulation in the tumor ∼ 6% ID/g at the tumor and drastic reduction of kidney uptake at 5% ID/g at the kidneys. Therefore, the fusion of B4 to serum albumin binder alone contributed to increase of the tumor to kidney ratios from 0. 2 to 2. We further confirmed that the use of amino acid linker between chelator and DBCO resulted in 2-fold reduction in the overall dose in the kidneys. Conclusions: We demonstrated that systematic ADME optimization enables nanobodies as potential carriers of radioactive payloads. This framework is readily transferable to other nanobodies within the same class. Moreover, nanobodies optimized for radiopharmaceuticals can be extended to different types of payloads, including chemotherapeutic conjugates. Citation Format: Yogindra Vedvyas, Nathaniel R. Fredette, Yoo-Shin Kim, Yang Yanping, Benedict Law, Moonsoo Jin. Rational engineering of nanobody pharmacokinetics and linker chemistry to maximize targeted radiotherapy efficacy abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (8Suppl): Abstract nr LB470.
Vedvyas et al. (Fri,) studied this question.