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. Author manuscript; available in PMC: 2021 Jul 15.
Published in final edited form as: Clin Cancer Res. 2020 Sep 21;27(2):532–541. doi: 10.1158/1078-0432.CCR-20-2150

A Self-Assembling and DisAssembling (SADA) bispecific antibody (BsAb) platform for curative 2-step pre-targeted radioimmunotherapy

Brian H Santich 1, Sarah M Cheal 2, Mahiuddin Ahmed 1, Michael R McDevitt 2,3,4, Ouathek Ouerfelli 5, Guangbin Yang 5, Darren R Veach 2, Edward K Fung 3, Mitesh Patel 2, Daniela Burnes Vargas 2, Aiza A Malik 1, Hong-Fen Guo 1, Pat B Zanzonico 6, Sebastien Monette 7, Adam O Michel 7, Charles M Rudin 2, Steven M Larson 2,3, Nai-Kong V Cheung 1,*
PMCID: PMC7855367  NIHMSID: NIHMS1636668  PMID: 32958698

Abstract

Purpose:

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

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 x anti-DOTA). SADA-BsAbs were assessed with multiple in vivo tumor models using 2-step pretargeted radioimmunotherapy (PRIT) to evaluate tumor uptake, dosimetry and anti-tumor 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, 2-step SADA-PRIT safely delivered massive doses of alpha-emitting (225Ac, 1.48 MBq/kg) or beta-emitting (177Lu, 6,660 MBq/kg) 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. Due to its modularity, SADA-BsAbs can be easily adapted to most tumor antigens, tumor types, or drug delivery approaches to improve TI and maximize the delivered dose.

Keywords: pretargeting, cancer, radioimmunotherapy, theranostic, therapeutic index

Introduction

Metastatic disease remains a major challenge to cancer cures (1). While localized disease can be controlled by surgery or radiation therapy, widespread, distant and occult metastases require systemic therapies (2). However, many of these treatments have unintended dose-limiting toxicities to vital organs due to poor therapeutic indices (TI, the ratio of total drug uptake in the tumor uptake compared to other normal tissues)(3), which results in many patients receiving subtherapeutic doses of treatment followed invariably by tumor relapse. Even with tumor-specific targets, conventional 1-step delivery systems typically have TI below 10:1(4).

Multi-step targeting strategies (5), where tumor targeting agents (e.g. anti-tumor IgG) are delivered separately from the cytotoxic payloads (e.g. chelated radioisotopes) have improved TIs, but dose-limiting toxicities remain. In conventional 2-step pre-targeted radioimmunotherapy (PRIT) the use of long-lived IgG molecules often results in myelotoxicity due to unbound IgG capturing and circulating the radioisotope (6) (Figure 1A). Although specially designed clearing agents can be used to remove this IgG, the resulting 3-step PRIT becomes substantially more complicated to translate to the clinic because the optimal clearing agent dose must be carefully titrated to individual tumor size and antigen density (7): administering too little risks insufficiently clearing the IgG, while administering too much risks interfering with payload uptake at the tumor (8). To safely maximize the administered dose in a clinically feasible way, the delivery strategy must use a 2-step approach (no clearing agent) with a tumor-targeting platform that can clear itself from the blood, bone marrow, liver, kidneys and other tissues.

Figure 1 –

Figure 1 –

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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).

In this study, we engineered such a platform using specially designed Self-Assembling and DisAssembling (SADA) domains (Figure 1B). When fused to bispecific antibodies (BsAb), the resulting SADA-BsAbs self-assembled into stable tetrameric complexes (220 kDa) that bound tumors with high avidity but could also disassemble into small dimers (110 kDa) or monomers (55 kDa) after a period of circulation in the blood (hours). Importantly, while the tetrameric complex exceeded the molecular weight (MW) cut off for renal filtration, the small monomers fell below the threshold (< ~70 kDa) and were rapidly and completely cleared from the blood. When combined with alpha- or beta-emitting radioisotopes, 2-step SADA-PRIT delivered massive doses of radiation (225Ac 1.48 MBq/kg, 177Lu 6,660 MBq/kg) to established solid tumors in multiple mouse models and ablated them without any clinical or histologic toxicities to the bone marrow, liver, kidneys, spleen, or brain.

Materials and Methods

Protein expression, reagents and binding studies

SADA proteins were expressed using the Expi293 Expression System (Invitrogen) and purified by affinity chromatography (GE, Ni-NTA Gravitrap, Cat# 11003399). S-2-(4-Aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA, ~580g/mol) was purchased from Macrocyclics and DOTA[175Lu]-thiourea-PEG4- 1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid[] (Proteus, ~1,350g/mol) was synthesized at Memorial Sloan Kettering according to previously described methods (9). Surface plasmon resonance (GE, Biacore T200) was performed as previously described (10).

Radiometal labeling

DOTA was labeled with 86Y nitrate (Radiological Chemistry and Imaging Laboratory at Washington University in St. Louis) or 177LuCl3 (Perkin Elmer) using previously described methods (2). Proteus was labeled with 225Ac nitrate (Oak Ridge National Laboratory) as described previously (9). Radioisotopes were given 48 hours after P53-SADA-BsAb in every study.

Animal studies

Greater detail on radiolabeling, pharmacokinetic studies and histological analyses are provided in the supplementary information. All animal experiments were performed in compliance with an Institutional Animal Care and Use Committee-approved protocol (protocol 09-05-010). Unless otherwise stated, BsAb doses were administered retroorbitally and all tumors were implanted subcutaneously on their right flank. Weights and tumor volumes were measured once per week, and overall mouse health was evaluated at least three times per week. Mice were sacrificed once tumor volumes reached 1,500-2,000 mm3. Animal experiments used either C57BL/6J (Jackson Laboratories, Cat# 000664), athymic nude (Envigo), NOD Prkdcscid IL2rgc−/− (NSG, Jackson Laboratories, Cat# 005557) or Balb/c Rag2−/− IL2rgc−/− mice (BRG, Taconic, Cat# 11503). All mice from the same treatment groups were co-housed in the same cage. Experiments using female mice were completely randomized before their initial treatment. Experiments using male mice had cages randomized before the start of treatment. Blinding of treatment or experimental measurements was not carried out.

Cell lines and tumor models

In vitro experiments were performed on a luciferase transfected IMR32 neuroblastoma cell line. In vivo studies used either IMR32 (ATCC CCL-127) or patient derived xenograft (PDX) tumors obtained from neuroblastoma or small cell lung cancer surgical specimens.

Pharmacokinetic analysis

Serum concentrations of BsAbs were determined by ELISA as previously described (11). Pharmacokinetic analysis was carried out by non-compartmental analysis of the serum concentration-time data using WinNonlin software program (Pharsight Corp.).

PET/CT Imaging analysis

Female nude mice (Hsd:athymic Nude-Foxn1nu 069(nu)/070(nu/+)) were implanted with IMR32 neuroblastoma tumors on day 0 and treated with BsAb and DOTA[86Y] (3.7 MBq, 30 pmol) on days 16 and 18, respectively. Mice treated with 3-step IgG-scFv-BsAb (8) received 25 μg of a DOTA-dendrimer clearing agent (7) four hours prior to the administration of DOTA[86Y]. Mice were imaged using PET/CT (Siemens, Inveon PET/CT) 16 hours later, with PET data collected for 30 minutes (a minimum of 1x106 coincidence events) followed whole-body CT scans acquired with a voltage of 80 kV and 500 μA. Image data were normalized to correct for nonuniformity of response of the PET, dead-time count losses, positron branching ratio, and physical decay to the time of injection but no attenuation, scatter, or partial-volume averaging correction was applied.

Tissue biodistribution analysis and dosimetry

Female nude mice were implanted with IMR32 tumors on day 0 on their right flank. Established tumors were treated with BsAb on day 16 and DOTA[177Lu] (1.85 to 18.5 MBq) on day 18. At 2, 24, 48, or 120 hours after administration of DOTA[177Lu], tissues were excised for gamma scintillation counting (Perkin Elmer). Dosimetry estimates were modeled using the non-decay-corrected time-activity concentration data fit to a 1-component, 2-component, or more complex exponential function as appropriate, and analytically integrated to yield the accumulated activity concentration per administered activity (MBq-h/g per MBq). The 177Lu equilibrium dose constant for non-penetrating radiations (8.49 g-cGy/MBq-h) was used to estimate the tumor-to-tumor and select organ-to-organ self-absorbed doses, assuming complete local absorption of the 177Lu beta rays only and ignoring the gamma ray and non-self-dose contributions.

Immunogenicity Analysis

C57BL/6J mice were injected with P53-SADA-BsAb or IgG-scFv-BsAb (0.5 nmol) on days 0 and 28, intravenously and intraperitoneally, respectively. Plasma were obtained on days 27 and 55. Anti-drug antibody (ADA) concentrations were determined by ELISA. Wells were coated with the corresponding BsAbs, blocked with a bovine serum albumin (0.5% in PBS) and incubated with plasma samples in duplicates. Binding was detected using an HRP-conjugated goat anti-mouse antibody (Jackson ImmunoResearch) and developed with o-phenylenediamine (Sigma). For quantitation, standard curves were generated using either mouse-anti-HIS (P53-SADA-BsAb) or mouse-anti-human IgG-hinge (IgG-scFv-BsAb) antibodies. Absorbance was measured at 490 nm (Synergy) and concentrations were estimated using GraphPad Prism 8.

Treatment studies

Radiation studies were performed on athymic nude mice or BRG mice (C.Cg-Rag2tm1Fwa Il2rgtm1Sug/JicTac) (12). Mice were implanted with tumors on their right flank between 12 and 18 weeks of age. Established solid tumors were treated at 14-20 days later with BsAb and either Proteus[225Ac] (37 kBq) or DOTA[177Lu] (18.5-55.5 MBq).

Anatomic and Clinical Pathology for Toxicology Assessment

Acute hematologic toxicities were assessed by automated CBC analysis (Drew Scientific) or cytokine FLT3L ELISA (RND systems, Cat# MFK00) according to manufacturer guidelines. Long-term toxicities were determined by complete necropsy and subsequent clinical or histological analysis, with age-matched littermates for reference. Serum chemistry was evaluated on an AU 680 chemistry analyzer (Beckman Coulter Inc), see supplementary tables 6 and 7 for a complete list of analytes. Hematology was measured by automated CBC analysis (Idexx laboratories Inc.) and validated by manual differential. Tissues were fixed in formalin and embedded in paraffin blocks, sectioned at 5 microns, stained with hematoxylin and eosin (H&E), and examined by board-certified veterinary pathologists (See supplementary information for a complete list of tissues).

Statistical analyses

All statistical analyses were performed using Graphpad Prism version 8.4 . Statistical significances were determined by Mann-Whitney tests (ADA titers), two-way analysis of variance (ANOVA) with subsequent Sidak correction (tumor responses), or a Log-rank Mantel-Cox test (survival analyses). For all statistical tests, a P value of < 0.05 was used to denote statistical significance.

Results

P53-SADA-BsAbs form stable tetramers with improved binding avidity

Previous reports have demonstrated that the optimal size for tumor penetrating proteins is between 150 and 200 kDa (13), while the renal clearance threshold has been estimated to be approximately 70 kDa (14). Based on this range, we designed a novel platform that fused small tetramerizing SADA domains to a humanized tandem single-chain fragment (scFv) BsAb: one scFv against ganglioside GD2 (15) and one scFv against radiometal-bearing DOTA (16). Candidate SADA domains were selected on several criteria – from the human proteome, soluble (non-membrane), naturally tetramerizing, and small (< 15 kDa) – to create SADA-BsAbs that would be ~200 kDa when self-assembled and ~50 kDa when disassembled (Figure 1B). Among the candidates identified (Supplementary Table 1–2), P53-SADA-BsAb was deemed the most promising for clinical development. Binding studies using surface plasmon resonance (GE, Biacore) confirmed P53-SADA-BsAb bound GD2 multivalently (Figure 2A), displaying a slower dissociation rate and increased apparent binding affinity (1.2 nM KD) compared to the corresponding anti-GD2 IgG-[L]-scFv formatted BsAb (IgG-scFv-BsAb, 4.6 nM KD)(8).

Figure 2 –

Figure 2 –

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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).

P53-SADA-BsAbs rapidly clear from the body without compromising tumor uptake

Serum pharmacokinetic analysis was performed on tumor-free NSG mice and demonstrated a 9-hour terminal half-life for P53-SADA-BsAb, with complete clearance from the blood within 48 hours (Supplementary Table 3). Notably, this was substantially slower than monomeric and dimeric anti-GD2 tandem-scFv BsAb (t1/2 = 0.5 hour)(17), while also faster and more complete than anti-GD2 IgG or IgG-scFv-BsAb (t1/2 = 72 hours)(10). Tumor uptake was measured using 2-step SADA-PRIT in athymic nude mice xenografts. Mice were dosed with P53-SADA-BsAb (1.25 nmol) and either 3.7, 18.5 or 37 MBq of DOTA[177Lu] (20, 100, or 200 pmol, respectively). Tumor uptake revealed a strong linear correlation with administered dose (slope of 0.45 pmol/g/MBq, R2 = 0.94), while activity in the blood remained low at all dose levels (slope < 0.001, R2 = 0.70), resulting in higher tumor to blood ratios with higher doses of payload (Figure 2B, Pearson coefficient of 0.9939). Kidney uptake also increased with administered dose but with a much shallower slope (slope = 0.04, R2 = 0.77). In contrast to previous studies of 3-step IgG-PRIT (2), where tumor uptake plateaued at about 11 pmol/g, these results suggest that P53-SADA-BsAb may be able to more effectively deliver DOTA payloads to the tumor.

Dosimetry estimates for 2-step SADA-PRIT were generated through serial biodistributions using the same animal model (Supplementary Table 4). Based on these findings, P53-SADA-BsAb was estimated to safely deliver an absorbed dose of 5,000 cGy to the tumor using a 15 MBq dose of DOTA[177Lu] payload, with the kidneys and blood receiving only 191 cGy and 44 cGy, respectively. Based on the linear dose-uptake relationship described above, further optimization should allow for continued improvements in these therapeutic indices.

To demonstrate the theranostic potential of P53-SADA-BsAb, we imaged mice using positron emission tomography (PET) (Figure 2C). Tumor bearing athymic nude mice were dosed with P53-SADA-BsAb at t=0h, followed by DOTA[86Y] at t=48h, and imaged at t=66h (Supplementary Videos 1–3). To accurately quantitate tissue uptake, mice were sacrificed, dissected, and counted directly after imaging (Supplementary Table 5). For comparison, two additional groups were included: mice dosed with (i) IgG-scFv-BsAb and DOTA[86Y] without clearing agent (2-step) or (ii) IgG-scFv-BsAb and DOTA[86Y] with clearing agent (3-step). As expected, 2-step IgG-scFv-BsAb (no clearing agent) demonstrated significant retention of DOTA[86Y] payload in the blood, while 3-step IgG-scFv-BsAb (with clearing agent) improved tumor contrast but also increased gut uptake. However, 2-step P53-SADA-BsAb (without clearing agent) displayed the best contrast, with strong tumor uptake (31 %ID/g) and almost no detectable signal in any other organ (0.1 %ID/g in blood, 0.8 %ID/g in liver, %0.3-0.8 ID/g in gut).

P53-SADA-BsAbs are much less immunogenic than IgG-scFv-BsAbs

Given their rapid clearance, we hypothesized P53-SADA-BsAbs may be less immunogenic than IgG-based therapies by avoiding the development of anti-drug antibody (ADA). Such a benefit would be critical to clinical translation, where multiple doses of antibody might be needed. To test this hypothesis, we immunized (day 0) and challenged (day 28) immunocompetent tumor-free mice with P53-SADA-BsAb or IgG-scFv-BsAb, and measured ADA titers in the plasma (Figure S1). Despite their high sequence homology, mice immunized with P53-SADA-BsAb showed significantly lower ADA titers than mice immunized with IgG-scFv-BsAb after both primary and secondary immunizations (P = 0.008).

P53-SADA-BsAb safely deliver beta-emitter payloads to ablate established neuroblastoma tumors

The anti-tumor function of 2-step SADA-PRIT was evaluated using a conventional 3x-3x schedule, where each week for three weeks, tumor bearing athymic nude mice received one dose of BsAb (1.25 nmol) was followed by one dose of DOTA[177Lu] (18.5 MBq, 100 pmol) 48 hours later (Figure 3A). Within two weeks all treated tumors had decreased in size, and within five weeks they had completely responded (Figure 3B), extending survival significantly (Figure 3C, median survival >115 d vs 20 d for control groups, P < 0.0001). After up to 8 months of follow-up, 70% (7/10) for P53-SADA-BsAb treated mice remained in complete remission.

Figure 3 –

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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.

Treatment-related toxicities stemming from P53-SADA-BsAb were determined after both short-term (0-30 days) and long-term (3-8 months) follow-up (Figures S2, Supplementary Table 6) and compared to 3-step IgG-PRIT using the corresponding IgG-scFv-BsAb (8). Overall, toxicities were mild or absent in P53-SADA-BsAb treated mice: there was no reduction in body weight, CBCs, plasma levels of cytokine FLT3L (a marker of bone marrow damage in mice and humans) (18,19) and serum chemistries were all normal, and histological analyses of the bone marrow, liver, kidney, spleen, brain and spine revealed no treatment-related pathologies. Two treatment-related toxicities were observed, however. One mouse treated with P53-SADA-BsAb (out of 9 checked) displayed mild to moderate ovarian atrophy (Figure S2), however this was substantially less common and less severe than the ovarian toxicity observed in 3-step IgG-PRIT (7/9 mice). In addition, one mouse (out of 9 checked) displayed chronic cystitis of the bladder (Figure S2), an observation also made in the 3-step IgG-PRIT treated mice (1 of 9 mice), suggesting that the toxicity stemmed from DOTA[177Lu] itself (which clears into the urine) and not the P53-SADA-BsAb.

2-step SADA-PRIT ablates established neuroblastoma PDX tumors using 177Lu

Based on the improved tumor dosimetry with larger doses of DOTA[177Lu], we evaluated the impact of delivering a 3-fold more DOTA[177Lu] per dose (55.5 MBq/dose, 300 pmol) using BRG mice bearing neuroblastoma PDXs (Figure 4A). All treated mice demonstrated complete and durable responses (5/5 mice) while control groups had to euthanized within 30 days due to tumor burden (Figure 4B–C).

Figure 4 –

Figure 4 –

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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.

Consistent with the previous model, P53-SADA-BsAb treated mice did not display any toxicity to the bone marrow, liver, kidney, spleen, brain or spine (Figure S3, Supplementary Table 7), however, mice did display a more severe cystitis of the bladder. Since these mice received 3-fold more radioisotope than the previous model (Figure 2B), the increased frequency and severity of the toxicity suggested that this amount of activity (6,600 MBq/kg 177Lu) was approaching the maximum tolerable dose to the bladder. Notably, serum chemistries for all treated mice were normal at the time of sacrifice, and the mice did not show any signs of overt urinary dysfunction during or after treatment, indicating that this toxicity likely developed slowly over many weeks.

2-step SADA-PRIT ablates established neuroblastoma PDX tumors using 225Ac

We recently developed a novel chelator (Proteus) for delivering 225Ac (9), which uses a non-radioactive DOTA[175Lu] handle for recognition by our anti-DOTA antibody and sought to evaluate it using 2-step SADA-PRIT. Here, BRG mice bearing the same neuroblastoma PDX tumors were treated with only one cycle of P53-SADA-BsAb (1.25 nmol) and Proteus[225Ac] (37 kBq, 2.4 nmol) (Figure 5A). All mice treated with P53-SADA-BsAb and Proteus[225Ac] responded, including one mouse which had a tumor over 500 mm3 in size (Figure 5A–C), but no toxicities were observed (Figure S4, Supplementary Table 7). CBC analysis confirmed no acute myelosuppression and serum chemistry values remained normal 120 days after treatment. In addition, histologic examinations of all tissues revealed no evidence of radiation damage. Notably, bladder toxicity was entirely absent, confirming that the cystitis observed previously (Figure S3) came from the specific radioisotope used (177Lu, perhaps due to its longer path-length) and not the P53-SADA-BsAb itself. Many previous studies using 225Ac have been limited by renal toxicity (20–22), and since SADA-BsAb cleared primarily by renal filtration, kidneys from treated mice were more thoroughly analyzed for histological changes. No treatment related pathologies were observed by either H&E, TUNEL staining, or cleaved caspase-3 (CC-3) immunohistochemistry, confirming that 2-step SADA-PRIT completely spared the kidneys.

Figure 5 –

Figure 5 –

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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.

P53-SADA-BsAb can ablate established small-cell lung cancer PDX tumors

Ganglioside GD2 is expressed in a broad spectrum of human tumors besides neuroblastoma (23). Among them, small-cell lung cancer (SCLC) is perhaps the most difficult to treat (5-year survival of < 5%) (24) and is currently undergoing clinical testing with an anti-GD2 IgG (NCT03098030). To evaluate its response to PRIT, we treated SCLC PDX (25) bearing BRG mice (Figure 6) with a single cycle of P53-SADA-BsAb (1.25 nmol) and Proteus[225Ac] (37.5 kBq, 700 pmol). Despite their massive size at the time of treatment, all treated tumors responded. Additionally, all but one tumor, the largest among them, shrank completely and durably, while tumors in control groups rapidly grew out to the maximum allowed sizes.

Figure 6 –

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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.

Discussion

In this study, we demonstrated that SADA domains can alter the pharmacokinetics of antibodies and sharply improve their therapeutic indices, a major hurdle for antibody or peptide-targeted therapies. While many protein therapies benefit from long terminal half-lives (26), the delivery of highly cytotoxic payloads is inevitably dose-limited by such platforms (27) and consequently this reduces anti-tumor efficacy. However, rapidly clearing peptides are not the solution either, due to their reduced bioavailability, poor tumor uptake and high kidney retention (28). The SADA platform takes advantage of the narrow pharmacokinetic window between these two approaches, allowing for a plasma half-life just long enough to reach the tumor, and just short enough to be completely removed from the blood before payload administration. Furthermore, achieving this pharmacokinetic window resulted in substantially reduced immunogenicity, an important consideration for any therapeutic strategies that require multiple treatment cycles.

Previous attempts with multimerized delivery platforms have failed to achieve the desired TI because they lacked critical attributes unique to our design: (i) the use of human domains (P53) that avoided the immunogenicity or kidney retention problems seen with streptavidin (29); (ii) the monomer size (55 kDa) that allowed both the full tetramer (220 kDa) and dimer intermediates (110 kDa) to exceed the renal threshold, in contrast to previous studies where smaller monomers (30 kDa) resulted in dimers (60 kDa) that fell below renal filtration threshold (30); and (iii) the use of a DOTA payload that has minimal non-specific uptake (31), in contrast to more hydrophobic haptens or peptides that are retained in the kidneys or other tissues (32).

Although treatment with P53-SADA-BsAb did not cause damage to any of the commonly radiosensitive tissues (bone marrow, liver, kidneys, spleen)(33–35), or natively GD2-expressing tissues (brain, spine)(36), two notable toxicities were observed in this study. Cystitis of the bladder in mice treated with DOTA[177Lu] appeared to be dose-dependent and related to the transit of 177Lu through the bladder, but not P53-SADA-BsAb itself (no toxicity was observed with 225Ac). Thus it is conceivable that any bladder damage could be minimized or even prevented with continuous emptying of the bladder (e.g. catherization, diuresis), or radioprotectants. In addition, some ovarian toxicity was observed, although substantially less often and less severely than among mice treated with conventional RIT or 3-step IgG-PRIT (33). Both toxicities were observed in tissues not routinely examined during preclinical studies, making it difficult to know how common they may be in other approaches. Future studies, especially when using radiation, should therefore include complete histologic analyses to uncover these unanticipated toxicities before they reach patients, especially for pediatric indications.

In conclusion, SADA has proven itself as a robust platform for the safe and effective targeted delivery of radioisotopes. It was efficacious without the need for clearing agent, and successfully treated two different aggressive solid tumors: neuroblastoma and small cell lung cancer. Given the striking lack of toxicity in the bone marrow, kidneys, or liver, SADA could provide substantial improvements in TI with other tumor targets or payloads, dramatically increasing anti-tumor efficacy while eliminating acute or long term toxicities.

Supplementary Material

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Translational Relevance:

Over 90% of cancer therapeutics currently in clinical development will fail, mostly due to dose-limiting toxicities or subtherapeutic recommended phase two dose levels, a consequence of insufficient therapeutic index (TI). We have developed a novel drug delivery platform (SADA) with unique pharmacokinetics that allowed for dramatic improvements in TI and substantially reduced immunogenicity. As a diagnostic, SADA visualized tumors with high precision using PET. As a therapeutic, SADA durably ablated two aggressive solid tumors (small cell lung cancer and neuroblastoma) using 177Lu or 225Ac, without any toxicity to the bone marrow, liver, or kidney. To the best of our knowledge, this is the first example of a curative radioimmunotherapy strategy without such toxicities. Furthermore, SADA is highly modular and can be adapted to improve many other drug delivery systems. Based on the findings presented here, the SADA platform has entered development for first-in-human clinical trials.

Acknowledgments:

We thank Dr. Elisa de Stanchina for her help in developing and passaging the patient-derived xenograft tumors. We also thank Rebecca Wilen for help with protein expression.

Funding: This work was supported by funds from the Isabella Santos Foundation (NKC), the Jeff Gordon Children’s Foundation (NKC), Enid A. Haupt Endowed Chair (NKC), the Robert Steel Foundation (NKC), the Grayer Fellowship (BS), Experimental Therapeutics Center of MSKCC (SML), R01 CA233896 (SMC), the MSKCC Core Grants (P30 CA008748) and a sponsored research agreement from Y-mAbs Therapeutics.

Disclosure of Potential Conflicts of Interest: BS, SMC, MA, MM, OO, SML and NKC were named as inventors on licensed and unlicensed patents filed by MSK. SADA has been licensed to YmAbs. NKC received sponsored research grants from Y-mAbs, had financial interest in Y-mAbs, Abpro-Labs, and Eureka Therapeutics, and is a SAB member for Abpro-Labs and Eureka Therapeutics. SML received commercial research grants from Genentech, Inc., WILEX AG, Telix Pharmaceuticals Limited, and Regeneron Pharmaceuticals, had financial interest Elucida Oncology Inc. and ImaginAb, Inc. SML consulted for Cynvec LLC, Eli Lilly & Co., Prescient Therapeutics Limited, Advanced Innovative Partners, LLC, Gerson Lehrman Group, Progenics Pharmaceuticals, Inc., and Janssen Pharmaceuticals, Inc. OO is an unpaid SAB member with financial interest in Angiogenex. MM consulted for Actinium Pharmaceuticals, Regeneron, Progenics, Bridge Medicine and General Electric. CMR consulted for AbbVie, Amgen, Ascentage, AstraZeneca, BMS, Celgene, Daiichi Sankyo, Genentech/Roche, Ipsen, Loxo and PharmaMar and is on the SAB of Elucida, Bridge and Harpoon. MA has financial interest in Y-mAbs. The other authors declare they have no competing interests.

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