A Self-Assembling and Disassembling (SADA) Bispecific Antibody (BsAb) Platform for Curative Two-step Pretargeted Radioimmunotherapy

Abstract

Purpose: Many cancer treatments suffer from dose-limiting toxicities to vital organs due to poor therapeutic indices. To overcome these challenges we developed a novel multimerization platform that rapidly removes tumor-targeting proteins from the blood to substantially improve therapeutic index.

Experimental design: The platform was designed as a fusion of a self-assembling and disassembling (SADA) domain to a tandem single-chain bispecific antibody (BsAb, anti-ganglioside GD2 × anti-DOTA). SADA-BsAbs were assessed with multiple in vivo tumor models using two-step pretargeted radioimmunotherapy (PRIT) to evaluate tumor uptake, dosimetry, and antitumor responses.

Results: SADA-BsAbs self-assembled into stable tetramers (220 kDa), but could also disassemble into dimers or monomers (55 kDa) that rapidly cleared via renal filtration and substantially reduced immunogenicity in mice. When used with rapidly clearing DOTA-caged PET isotopes, SADA-BsAbs demonstrated accurate tumor localization, dosimetry, and improved imaging contrast by PET/CT. When combined with therapeutic isotopes, two-step SADA-PRIT safely delivered massive doses of alpha-emitting (²²⁵Ac, 1.48 MBq/kg) or beta-emitting (¹⁷⁷Lu, 6,660 MBq/kg) S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) payloads to tumors, ablating them without any short-term or long-term toxicities to the bone marrow, kidneys, or liver.

Conclusions: The SADA-BsAb platform safely delivered large doses of radioisotopes to tumors and demonstrated no toxicities to the bone marrow, kidneys, or liver. Because of its modularity, SADA-BsAbs can be easily adapted to most tumor antigens, tumor types, or drug delivery approaches to improve therapeutic index and maximize the delivered dose.See related commentary by Capala and Kunos, p. 377.

Figure 1 –

Overview of multi-step radioimmunotherapy (A) Schematic of 4 different radioimmunotherapy strategies. Representative antibodies, radioisotope payloads, and clearing agent are included for reference. Blue antibody domains are tumor-specific, and orange domains are DOTA-specific. The red curve represents concentration of radioisotope in the blood over time, blue represents the concentration of radioisotope in the tumor and grey represents the concentration of non-radioactive antibody in the blood. (B) Representative P53-SADA-BsAb. Each monomer (inset) is made of 3 domains: an anti-tumor domain (blue), an anti-DOTA domain (orange) and a SADA domain (purple), from N-terminus to C-terminus, respectively. SADA domains self-assemble into tetramers (~200 kDa) but also disassemble into monomers (~50 kDa).

Figure 2 –

In vivo pharmacokinetics and biodistribution of P53-SADA-BsAb (A) Normalized GD2 binding kinetics of P53-SADA-BsAb compared to IgG-scFv-BsAb, as measured by SPR. For each curve maximum binding was normalized to 100. (B) Relationship between administered dose and tissue uptake using 2-step SADA-PRIT. Mice (n=5 per group) were administered P53-SADA-BsAb (1.25 nmol) and one of 3 doses of DOTA[¹⁷⁷Lu]: 3.7, 18.5 or 37 MBq (20, 100 or 200 pmol, respectively). Blue represents level of DOTA payload in the tumor, green represents the kidney, and red represents the blood. The purple points represent the therapeutic index between tumor and blood at each dose. Tissue uptake was normalized to pmol of DOTA[¹⁷⁷Lu] per gram of tissue. (C) PET/CT using P53-SADA-BsAb. Representative schematic (left) and images (right). Mice (n=1-2) were injected with P53-SADA-BsAb or IgG-scFv-BsAb (with and without clearing agents) followed by DOTA[⁸⁶Y] (green lines correspond to each injection). Mice were imaged for 30 minutes (grey arrow) 18 hours after the administration of DOTA. Representative images are normalized using the same scale. Orange arrows point to the subcutaneous tumor (left panel) or the bladder (middle panel).

Figure 3 –

Neuroblastoma xenograft treatment study (A) Schematic of treatment model (left) and mean tumor responses (right). Each dose of BsAb (1.25 nmol, triangle) was followed by one dose of DOTA[¹⁷⁷Lu] (18.5 MBq, star) 48 hours later. Each solid line represents one treatment group (n=10). The dotted black line represents no measurable tumor, and the orange hexagon represents the tumor implantation. Tumor averages were calculated until at least one mouse had to be euthanized. (B) Individual tumor responses. Each solid line represent tumors from a single mouse, and the dashed line represents the group average. (C) Progression-free survival analysis. Each mouse was measured until tumors grew above 500 mm³ in size. Mice were also censored for clinical pathology. No mice died unexpectedly. **P < 0.01, ****P < 0.0001 using two-way ANOVA (with Sidak correction) or Log-rank (Mantel-Cox) test.

Figure 4 –

Neuroblastoma PDX treated with DOTA[¹⁷⁷Lu] (A) Schematic of DOTA[¹⁷⁷Lu] treatment model (left) and mean tumor responses (right). Each dose of BsAb (1.25 nmol, triangle) was followed by one dose of DOTA[¹⁷⁷Lu] (55.5 MBq, star) 48 hours later. Each solid line represents one treatment group (n=5). The dotted black line represents no measurable tumor, and the orange hexagon represents the tumor implantation. Tumor averages were calculated until at least one mouse had to be euthanized. (B) Individual tumor responses. Each solid line represent tumors from a single mouse, and the dashed line represents the group average. (C) Progression-free survival analysis. Each mouse was measured until tumors grew above 500 mm³ in size. No mice died unexpectedly. **P < 0.01, ****P < 0.0001 using two-way ANOVA (with Sidak correction) or Log-rank (Mantel-Cox) test.

Figure 5 –

Neuroblastoma PDX treated with Proteus[²²⁵Ac] (A) Schematic of Proteus[²²⁵Ac] treatment model (left) and mean tumor responses (right). Each dose of BsAb (1.25 nmol, triangle) was followed by one dose of Proteus[²²⁵Ac] (37 kBq, star) 48 hours later. Each solid line represents one treatment group (n=5). The dotted black line represents no measurable tumor, and the orange hexagon represents the tumor implantation. Tumor averages were calculated until at least one mouse had to be euthanized. (B) Individual tumor responses. Each solid line represent tumors from a single mouse, and the dashed line represents the group average. (C) Progression-free survival analysis. Each mouse was measured until tumors grew above 500 mm³ in size. No mice died unexpectedly. **P < 0.01, ****P < 0.0001 using two-way ANOVA (with Sidak correction) or Log-rank (Mantel-Cox) test.

Figure 6 –

Small-cell lung cancer PDX treatment study (A) Schematic of Proteus[²²⁵Ac] treatment model (left) and mean tumor responses (right). One dose of BsAb (1.25 nmol, triangle) was followed by one dose of Proteus[²²⁵Ac] (37 kBq, star) 48 hours later. Each line represents one treatment group (n=5). The dotted black line represents no measurable tumor, and the orange hexagon represents the tumor implantation. Tumor averages were calculated until at least one mouse had to be euthanized. (B) Individual tumor responses. Each solid line represent tumors from a single mouse, and the dashed line represents the group average. ****P < 0.0001. (C) Progression-free survival analysis. Each mouse was measured until tumors grew above 500 mm³ in size. No mice died unexpectedly. **P < 0.01, ****P < 0.0001 using two-way ANOVA (with Sidak correction) or Log-rank (Mantel-Cox) test.