CD38-SADA, a Self-Assembling and Dis-Assembling Bispecific Fusion Protein for Two-Step Pretargeted Radioimmunotherapy of Non-Hodgkin Lymphoma
Authors: Santich Brian H. , Wang Linlin , Abdulsalam Azeezat , Vadlamudi Mallika , Liebenberg Nico , Ouattara Amadou , Jani Kandarp , Nagel Johannes , LaFrance Norman , Russell Charlotte A. 
Published: December 2024

Background
Radioimmunotherapy (RIT) for the treatment of hematological malignancies has clarified the need for pretargeted RIT (PRIT) that delivers tumor-killing radiation while limiting exposure to healthy organs and tissues. CD38-SADA is a bispecific fusion protein that binds the CD38 antigen and select radioisotopes chelated to tetraxetan (“DOTA”). It also contains a p53-derived tetramerization domain that drives the self-assembling and disassembling of CD38-SADA tetramers, each with 4 distinct binding sites for CD38. In a pretargeting infusion, nonradiolabeled CD38-SADA tetramers bind to CD38-expressing tumor cells, while unbound CD38-SADA protein disassembles into renally cleared monomers. The second infusion administers the chelated therapeutic radioisotope, which binds to the CD38-SADA and irradiates the tumor cells. Here, we present the preclinical binding properties of CD38-SADA, and the anti-tumor efficacy of CD38-SADA PRIT with lutetium 177 (Lu177)-DOTA in a xenograft mouse model.
Methods
Binding affinities of CD38-SADA to CD38 and to lanthanide metal (Ln)-DOTA complexes (terbium [Tb], europium [Eu], lutetium [Lu], and lanthanum [La]), to trace metal (TM)-DOTA complexes (iron [Fe], copper [Cu], or zinc [Zn]), and to empty DOTA were measured by enzyme-linked immunosorbent assay (ELISA). CD38-SADA binding to CD38+ human lymphoma cell lines was investigated with flow cytometry (FC). In a xenograft mouse model, anti-tumor responses were evaluated across dose ranges of CD38-SADA and Lu177-DOTA. Briefly, immunocompromised R2G2 female mice were inoculated subcutaneously with Daudi human CD38+ cells and received 2 weekly cycles of a single dose of intravenous (IV) CD38-SADA followed by an IV dose of Lu177-DOTA 48 hours later (n=6-8 mice per group).
Results
Competition ELISA assays showed strong binding to Lu175-DOTA (IC50=1.32 nM) and other lanthanide metals (IC50 for Tb=0.98, La=1.37, and Eu=0.91 nM) and negligible interactions with empty DOTA and TM-DOTA complexes. In ELISA assays, CD38-SADA tetramers showed greater binding to CD38 than obligate monomers lacking a p53-derived tetramerization domain (EC50=1.14 and 6.13 nM, respectively). In the FC analysis, CD38-SADA bound strongly to CD38+ human lymphoma cell lines (EC50 values=0.6 nM [Maver; Jurkat E6-1]; 1.0 nM [Ramos]; 1.7 nM [Daudi]; 6.4 nM [HuT-78]) but not to CD38- cell lines (EC50&gt;1667 µM). In mice pretargeted with CD38-SADA (10 mg/kg), the anti-tumor response correlated with increasing levels of Lu177-DOTA (297.1±150.3 mm3 and 130.5±43.5 mm3 mean tumor volume [MTV] at 0.5 or 1 mCi/mouse, respectively) and significantly exceeded control injections with Lu177-DOTA (1 mCi/mouse; 1156.3±182.8 mm3) or CD38-SADA (10 mg/kg; 981.2±243.2 mm3) alone (P&lt;0.001). At fixed Lu177-DOTA levels (1 mCi/mouse), anti-tumor responses were dose-dependent across CD38-SADA doses (MTV 349.5±167.9 mm3, 100.5±57.1 mm3, and 33.75±30.7 mm3 at 5, 10, and 30 mg/kg, respectively) vs controls with either Lu177-DOTA (1 mCi/mouse; 1412.3±190.4 mm3) or CD38-SADA (30 mg/kg; 1425.5±258.7 mm3) alone (P&lt;0.001). Compared with controls, mean survival times of mice treated with 5 or 10 mg/kg CD38-SADA at fixed Lu177-DOTA doses (1 mCi/mouse) increased by 25 and 47 days, respectively, while radiation exposure at high levels of CD38-SADA (30 mg/kg) and Lu177-DOTA (1 mCi/mouse) resulted in excessive weight loss (&gt;20%) and per-protocol euthanasia.
Conclusions
CD38-SADA showed high-affinity binding in vitro to the CD38 antigen and Ln-DOTA complexes and negligible binding to TM-DOTA and empty DOTA, the latter used in excess to ensure complete chelation. Negligible binding to empty DOTA suggests the absence of competitive antagonism with the radioactive payload in vivo. CD38-SADA tetramers, compared in vitro with obligate monomers, demonstrated marked increases in binding to CD38 and bound strongly to an array of CD38+ lymphoma cell lines. Taken together, the data show that CD38-SADA forms tetramers with high avidity for CD38 and selectivity for chelated lanthanides with current and emerging applications in targeted radiotherapy. When used in PRIT with Lu177-DOTA, this bispecific fusion protein demonstrated robust and dose-responsive anti-tumor efficacy in a mouse xenograft model of CD38+ Daudi cells, further supporting the clinical development of CD38-SADA PRIT with Lu177-DOTA in Trial 1201 (NCT05994157).