# Successful repositioning of mertansine for improved chemotherapy by combining a polymer prodrug approach and PET imaging

Soizic Martin-Aubert, Kevin Avrillon, Nicolas Tournier, Alexandre Bordat, Vu Long Tran, Nada Ibrahim, Dimitri Kereselidze, Benoit Jego, Léa Potiron, Tanguy Boissenot, et al.

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## To cite this version:

Soizic Martin-Aubert, Kevin Avrillon, Nicolas Tournier, Alexandre Bordat, Vu Long Tran, et al. Successful repositioning of mertansine for improved chemotherapy by combining a polymer prodrug approach and PET imaging. Journal of Controlled Release, 2025, 378, pp.803-813. 10.1016/j.jconrel.2024.12.050. hal-04927661

HAL Id: hal-04927661

https://hal.science/hal-04927661v1

Submitted on 3 Nov 2025

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Successful repositioning of mertansine for improved
chemotherapy by combining a polymer prodrug approach
and PET imaging

Soizic Martin-Aubert,¹ Kevin Avrillon, ¹ Nicolas Tournier, ¹ Alexandre Bordat,² Vu Long Tran,¹ Nada Ibrahim,² Dimitri Kereselidze, ¹ Léa Potiron,² Nicolas Tsapis,³ Julien Nicolas, ³ Tanguy Boissenot,².*

Charles Truillet, ¹,*

¹ Paris-Saclay University, CEA, CNRS, Inserm, Multimodal Biomedical Imaging Lab (BioMaps), Orsay, France.

² Imescia, Université Paris-Saclay, 91400 Saclay, France

³ Université Paris-Saclay, CNRS, Institut Galien Paris-Saclay, 91400 Orsay, France

*co-corresponding authors: charles.truillet@cea.fr & tanguy.boissenot@gmail.com

Soizic Martin-Aubert (soizic.martin-aubert@universite-paris-saclay.fr)

Kevin Avrillon (kevinav@free.fr)

Nicolas Tournier (nicolas.tournier@cea.fr)

Alexandre Bordat (abordat@gmail.com)

Vu Long Tran (vl.tran@hotmail.com)

Nada Brahim (Nada.brahim@gmail.com)

Dimitri Kereselidze (dimitri.kereselidze@gmail.com)

Léa Potiron (lea.potiron@hotmail.com)

Nicolas Tsapis (nicolas.tsapis@cnrs.fr)

Julien Nicolas (julien.nicolas@universite-paris-saclay.fr)

Tanguy Boissenot (tanguy.boissenot@gmail.com)

Charles Truillet (charles.truillet@cea.fr)
<figure><img src="image_3.png" /></figure>

**Abstract**

Mertansine (DM1), a potent tumor-killing maytansinoid, requires conjugation to antibodies or incorporation into nanocarriers due to its high toxicity. However, these carriers often result in undesirable biodistribution, leading to rapid and long-term accumulation in the kidneys or liver and potentially increased toxicity. To overcome this limitation, we used the hydrophilic, biocompatible, and stealth properties of polyacrylamide (PAAm) as a scaffold to develop water-soluble PAAm-DM1 polymer prodrugs, leveraging PAAm's previous success in delivering paclitaxel via subcutaneous administration. To monitor distribution and predict efficacy, we have imparted Positron Emission Tomography (PET) imaging capabilities to well-defined PAAm-DM1 polymer prodrugs. Our studies demonstrated the same tumor accumulation and the same distribution of PAAm-DM1 in the main organs such as liver, kidneys muscle, regardless of delivery route (subcutaneous or intravenous). Interestingly, tumor accumulation of PAAm-DM1 was primarily driven by passive accumulation, as indicated by PET imaging, without significantly altering treatment efficacy. This suggests complex mechanisms, possibly involving immune system interactions by influencing notably the metabolism and clearance. To enhance therapeutic outcomes, we combined the polymer prodrug with immunotherapy, specifically anti-CTLA4. Our findings highlight the promising potential of PAAm-DM1, offering a novel formulation strategy for DM1 in cancer therapy.
Introduction

One of today's major challenges in cancer treatment is balancing the benefits of treatment with the associated risks. Chemotherapeutic drugs, although effective, lack selectivity and can damage healthy cells and tissues with rapid turnover rates, leading to severe toxic effects and, ultimately, the development of drug resistance.¹ The challenges associated with the nonspecificity and inadequate pharmacokinetics and pharmacodynamics of chemotherapeutic drugs are widely recognized.² Additionally, their bioavailability is often limited by their poor water solubility. In this context, benzoansamacrolide mertansine (DM1), a microtubulin inhibitor, is no exception. DM1 is a potent chemotherapeutic agent that binds to or close to the vinblastine-binding site in the mitochondrial region, leading to mitotic arrest and apoptosis-mediated cell death.³ Despite its promising antitumor activity, mertansine's toxicity, particularly in the gastrointestinal and nervous systems, has restricted its clinical translation to a solitary phase 1 clinical trial.⁴ Mertansine has always been administered under the form of antibody-drug conjugates (ADCs), such as cantuzumab or bivatuzumab, but with mixed results. In addition to their high cost, ADCs rely on a single biomarker for targeting, which can limit their broader applicability.

Polymer prodrugs offer a promising solution by administrating pharmacologically-inactive compounds that can be converted into active drugs via a simple chemical modification, particularly within the tumor microenvironment, where they can exert targeted therapeutic effects.⁵ Prodrugs primarily aim to enhance water solubility and chemical stability, enhancing bioavailability while also minimizing drug metabolism and toxicity.⁶ The challenge in formulating prodrugs lies in designing compounds encompassing these diverse characteristics while simultaneously maintaining or enhancing chemotherapeutic efficacy. Recently, we have developed a polymer prodrug approach by grafting polyacrylamide (PAAm), a hydrophilic and biocompatible polymer, to active pharmaceutical ingredients (API), such as paclitaxel (Ptx), in order to modify their physicochemical properties.⁷ This innovative strategy addresses several key limitations associated with the use of Ptx, particularly in terms of toxicity and solubility. Moreover, enhancing the solubility of Ptx enables its concentration within a smaller volume, which facilitates preparation and administration. Notably, PAAm surpasses all other water-soluble polymers available, demonstrating exceptional capability in solubilizing highly hydrophobic APIs. Furthermore, it leads to prolonged circulation time within the body and sustained release of the APIs, ensuring prolonged efficacy. It has also been shown that subcutaneous injection of PAAm-Ptx, Ptx being a representative hydrophobic irritant/vesicant anticancer drug, resulted in the prolonged release of Ptx into the bloodstream and greater anticancer efficacy than Taxol (the commercial formulation of Ptx), while avoiding local toxicity.

A number of questions have been raised, particularly regarding the distribution of the polymer prodrug in tumors and the possibility of specifically triggering the drug release at the tumor site, leveraging a
distinct passive accumulation within the tumor, particularly due to the enhanced permeability retention effect (EPR). This phenomenon, observed in solid tumors, facilitates the preferential accumulation of certain nanoscale objects, including nanoparticles, within tumor tissue. Considering the size of PAAm-API polymer prodrugs (around 20 kDa), understanding the potential influence of passive accumulation on tumoral distribution is crucial.⁸ Polymer-drug conjugates have already demonstrated efficient accumulation within tumors facilitated by passive accumulation.⁹ However, it's worth noting that the passive accumulation may vary among different patients and even within individual lesions.¹⁰

Several biomedical imaging techniques, including magnetic resonance imaging (MRI), ultrasound imaging, optical imaging, X-ray, computed tomography (CT), and nuclear imaging, have the potential to quantify the dynamic distribution of drug delivery. However, many of these techniques face limitations, such as sensitivity issues in MRI and CT, or spatial constraints in optical and ultrasound imaging.¹¹ In this context, Positron Emission Tomography (PET) imaging plays a key role in the advancement of oncological theranostics, which encompasses the study of drug distribution to target tissues and tumors for enhanced therapeutic management.¹²,¹³ PET imaging provides precise quantification of drugs, aiding in understanding tissue distribution and pharmacokinetics. Furthermore, PET imaging of the passive accumulation of nanoparticles has proven to be an effective predictive biomarker for the efficacy of nanoparticles drug delivery systems, thus stimulating the development of further PET imaging strategies to optimize efficacy.¹⁴,¹⁵

In this context, we devised a theranostic approach integrating PET imaging directly into the synthesis of water-soluble PAAm-based polymer prodrugs. This involves incorporating a chelate capable of binding with zirconium-89 (⁸⁹Zr), enabling imaging over a significant kinetic interval thanks to adequate half-life (3.3 days), consistent with the tissue kinetics of polymers. We investigated the bioavailability of PAAm-based polymer prodrugs with DM1 following both intravenous and subcutaneous injections, correlating tumor response in tumor models with PAAm uptake. We incorporated PET imaging to evaluate tumor drug exposure by correlating tumor accumulation with survival, aiming to personalize dosing, reduce toxicity, and extend applicability across a range of tumor types. To further amplify therapeutic outcomes, we combined the polymer prodrug with immunotherapy, specifically anti-CTLA4.

# Materials and methods

## Materials

[⁸⁹Zr]Zr-oxalic acid was purchased from PerkinElmer (Netherlands). Anti-mouse CTLA4, clone 9H10 was purchased from InvivoGen, Cat #BE0131. Fetal Bovine Serum (FBS). Most of chemical reagents including buffers were purchased at Sigma (Merk). CT26 and MC38 cell lines were purchased from the American Type Culture Collection and Kerafast respectively. For their culture, DMEM medium, RPMI-1640 medium, fetal bovine serum, antibiotic-antimycotic solution were purchased from Gibco.
Acrylamide (≥99%, AAm) was purchased from Sigma-Aldrich and recrystallized from chloroform. Azobisisobutyronitrile (98%, AIBN) was purchased from Sigma-Aldrich and recrystallized from ethanol. 4-Cyano-4-(((ethylthio)carbonothioyl)thio)pentanoic acid (95%, CEP) was purchased from Ambeed and used as received., N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (98%, EDC・HCl), Magnesium sulfate (99% anhydrous, MgSO4), N,N-diisopropylethylamine (99%, DIPEA), N-2(aminoethyl) carbamic acid-tert-butyl-ester (97%, N-Boc-ethylenediamine), triethylamine (99%, TEA), deferoxamine mesylate (95%, DFO), acrylic acid (99%) were purchased from ABCR and used as received. N-Hydroxysuccinimide (≥98%, NHS), Sodium Carbonate (99.5%, Na2CO3), Celite® 535, Hydrogen chloride 4 M in 1,4-dioxane (99%, HCl dioxane 4 M) were purchased from Thermofisher Scientific and used as received, 1-(2-aminoethyl)-2,5-dihydro-1H-pyrrole-2,5-dione hydrochloride (95%, AEMI) were purchased from Enamine and used as received. N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl) maytansine (DM1) and were purchased from DC Chemicals and used as received. Deuterium oxide (D2O), deuterated chloroform (CDCl3), deuterated Methanol (MeOD) and deuterated dimethyl sulfoxide (d6-DMSO) were obtained from Eurisotop. All solvents were purchased from Sigma-Aldrich at the highest grade.

## Cell culture

CT 26 and MC 38 cell lines are two colon carcinoma cell line presenting different common driver mutations leading to some difference in terms of immunogenicity.¹⁶ CT 26 were cultured in RPMI-1640 medium and MC 38 in DMEM medium, both supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution, at 37°C in a humidified atmosphere with 5% CO2.

## Animal experiments

Animal experiments were performed according to the European Directive 2010/63/EU and to its transposition into the French law (Decree No. 2013-118). The research project was conducted at the CEA-SHFJ imaging platform (authorization D91-471-105) and was approved by a local ethics committee (CETEA-CEA DSV IdF). Female Balb/c mice (Balb/cJrJ, Janvier, 5 weeks old when received) and female C57BI/6 (Janvier, 5 weeks old when received, 9 weeks old when the cells were implanted) were housed in individually-ventilated cages in a temperature (22°C) and humidity (40 %)-controlled room, with a 12 h light/12 h dark cycle. Animal experiments were performed under anesthesia with isoflurane in oxygen. Anesthetized mice were subcutaneously inoculated in both lower flanks respectively with CT 26 cells (10⁶ cells) or MC 38 cells (1.5x10⁶ cells) suspended in PBS (BD Biosciences).

For the two first experiments, female Balb/c mice (Balb/cJrJ, Janvier, 5 weeks old when received) were housed in individually-ventilated cages in a temperature (22°C) and humidity (40 %)-controlled room, with a 12 h light/12 h dark cycle. For the last experiment, female C57BI/6 (Janvier, 5 weeks old when received, 9 weeks old when the cells were implanted) were housed in the same conditions.
Animal experiments were performed under anesthesia with isoflurane in oxygen. Anesthetized mice were subcutaneously inoculated in both lower flanks with either CT 26 cells (10⁶ cells) or MC 38 cells (1.5x10⁶ cells) suspended in PBS (BD Biosciences).

## Synthesis of CEP-NHS

In a 250mL round-bottom flask, CEP (22.8 mmol, 6 g), EDC (22.9 mmol, 4.4 g) and NHS (26.9 mmol, 3.1 g) were dissolved in acetone (100 mL). The reaction was stirred 7h at room temperature. A white precipitate was observed, which was filtered and rinsed with water. The solid was then dissolved in a minimum amount of dichloromethane. The organic layer was evaporated and dried with MgSO4. The product was recrystallized from ethanol (350mL) to give a yellow powder. Yield: 87%, 1H NMR (300 MHz, CDCl3): δ 3.38 (q, *J* = 7.4 Hz, 2H), 3.05-2.78 (m, 6H), 2.77-2.45 (m, 2H), 1.93 (s, 3H), 1.40 (t, *J* = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 216.30, 168.72, 167.00, 118.60, 46.00, 33.70, 33.20, 31.42, 26.84, 25.55, 24.85, 24.75, 12.67. C₁₃H₁₆N₂O₄S₃ HRMS (M+H) +: 361.0345; found: 361.0350.

## Synthesis of CEP-AEMI-DM1

In a 100mL round-bottom flask, CEP (0.76 mmol, 200 mg), EDC (0.77 mmol, 147 mg) and NHS (0.96 mmol, 111 mg) were dissolved in acetone (18 mL). The reaction was stirred for 1h at room temperature. AEMI (0.77 mmol, 137 mg) and DIPEA (1.9 mmol, 246 mg) were dissolved in acetone (2 mL) and added to the reaction. The reaction was stirred for 3h at room temperature. DM1 (0.19 mmol, 140 mg) was dissolved in acetone (6 mL) and added to the reaction. The reaction was stirred for 3h supplementary at room temperature. The crude was purified on flash chromatography using a mixture of DCM/MeOH (92/8 v/v isocratic) as the mobile phase. The solvent was evaporated to yield a yellow powder. Yield: 73%. 1H NMR (300 MHz, CDCl3) δ 6.81 (d, *J* = 4.9 Hz, 1H), 6.71-6.61 (m, 2H), 6.44 (dd, *J* = 15.0, 11.0 Hz, 1H), 6.17 (s, 1H), 5.61 (dd, *J* = 14.0, 9.1 Hz, 1H), 5.27 (dd, *J* = 6.4, 2.8 Hz, 1H), 4.71 (dd, *J* = 11.7, 2.7 Hz, 1H), 4.39-4.16 (m, 3H), 3.96 (s, 3H), 3.80-3.54 (m, 3H), 3.46 (d, *J* = 8.8 Hz, 1H), 3.39-3.28 (m, 5H), 3.17 (s, 3H), 3.14-2.90 (m, 4H), 2.85 (s, 4H), 2.70-2.46 (m, 5H), 2.44-2.23 (m, 2H), 2.22-2.08 (m, 2H), 1.82 (d, *J* = 0.8 Hz, 3H), 1.72-1.56 (m, 5H), 1.40-1.14 (m, 11H), 0.92-0.84 (m, 3H). C₅₀H₆₇ClN₆O₁₃S₄ HRMS (M+H) +: 1105.3310; found: 1105.3329.

## Synthesis of AAm-DFO

In a 50 mL round-bottom flask, DFO mesylate (3 mmol, 2 g), EDC (3.9 mmol, 600 mg) and acrylic acid (8.5 mmol, 612 mg) were dissolved in anhydrous DMF (25 mL). Heat the mixture for 30 min at 45 °C. Then add DIPEA (12.9 mmol, 1666 mg). The reaction was stirred at room temperature overnight. The crude was purified by washing with diethyl ether (2 times 50 mL), a washing with diethyl ether (30 mL) and MeOH (3 mL) and a washing with water (2 times 50 mL). Filtrate and dry to give a white powder. Yield: 94%. 1H NMR (300 MHz, DMSO-d6) δ 9.78-9.48 (m, 3H), 7.84 (s,
2H), 6.31 – 6.13 (m, 1H), 6.13 – 5.97 (m, 1H), 5.57 (d, 2H), 3.73 – 3.16 (m, 6H), 3.16 – 2.88 (m, 4H), 2.67 – 2.38 (m, 2H), 2.38 – 2.15 (m, 4H), 1.97 (s, 3H), 1.64 – 1.05 (m, 16H). C₂₈H₅₀N₆O₉ HRMS (M+H) +: 615.3712; found: 615.3724.

## Synthesis of PAAm-Amide-DFO3%-DM1

To produce PAAm-Amide-DFO3%-DM1, in a vial, CEP-Aemi-DM1 (0.018 mmol, 20 mg), AIBN (0.073 mmol, 12 mg), Acrylamide (4.1 mmol, 291.1 mg) and AAm-DFO (0.13 mmol, 80 mg) were dissolved in DMSO (1.5mL). The mixture was degassed under argon for 15min and then let under agitation for 24h at 70°C. After cooling down to room temperature, the solution was precipitated in cold methanol under vigorous stirring to give beads. The beads were filtrated and redissolved in MilliQ water. The resulting solution was dialyzed in MilliQ water for 3 days. The solution was lyophilized for 3 days to give a white solid. Yield: 23%. Mn NMR 24.1kDa.

## Synthesis of PAAm-Amide-DM1 (PAAm-DM1)

Acrylamide (60 mmol, 4254 mg), CEP-Aemi-DM1 (0.22 mmol, 250 mg) and AIBN (0.05 mmol, 7.76 mg) were dissolved in DMSO. Argon was bubbled through the solution for 15min. The solution was stirred at 70°C for 2h. After cooling down to room temperature, the solution was precipitated in cold methanol under vigorous stirring to give beads. The beads were filtrated and redissolved in MilliQ water. The resulting solution was dialyzed in MilliQ water for 3 days. The solution was lyophilized for 3 days to give a white solid (3.6g, 84% conversion). Mn NMR 19.6kDa

## Nuclear Magnetic Resonance (NMR) Spectroscopy

1H NMR and 13C NMR spectroscopy of small molecules were performed in 5 mm diameter tubes in deuterated chloroform (CDCl3) on a Bruker Avance 300 spectrometer operating at 300 MHz (1H) or 75 MHz (13C) at room temperature. 1H NMR spectroscopy of polymers was performed on a Bruker Avance 3 HD 400 spectrometer operating at 400 MHz in 5 mm diameter tubes: (i) in d6-DMSO (70 °C, 128 scans) or in D2O (70 °C, 128 scans) for PAAm-based polymers; (ii) in D2O (70 °C, 128 scans) for DM1-PEG polymer. "Synthesis and Radiosynthesis of the [⁸⁹Zr]Zr-PAAm-DFO-DM1".

## Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS)

Liquid chromatography conditions were as follows: C18 (HILIC) column (Nucleodur, EC 125/2, 100-5-C18, Macherey-Nagel, Hoerdt, France). Mobile phase: acetonitrile/water (50/50, v/v) with formic acid 0.1%; run time: 8 min; flow rate: 0.3 mL min⁻¹. ESI-MS/MS analyses were performed on a triple quadrupole mass spectrometer detector (TQD) with an electrospray ionization (ESI) interface (Quattro Ultima, Waters, Guyancourt, France). Electrospray and mass parameters were optimized by direct infusion of pure analytes into the system. ESI parameters: capillary voltage of 3.5 kV, cone voltage of
35 V, source temperature of 120 °C, desolvation temperature of 350 °C, with a nitrogen flow of 506 L·h⁻¹. Supplementary information “Synthesis and Radiosynthesis of the [⁸⁹Zr]Zr-PAAm-DFO-DM1”.

## [⁸⁹Zr]Zr-PAAm-DFO-DM1 radiolabeling process

Radiolabeling with ⁸⁹Zr was performed according to a published protocol.¹⁷ Briefly, 170 µL of [⁸⁹Zr]Zr-oxalic acid solution was neutralized with Na₂CO₃ and incubated with 1.5 mL of PAAm-DFO-DM1 solution (4 mg/mL in PBS) for 1 hour at 37 °C. The mixture was adjusted to pH 7.2 with HEPES (1 M, pH 7.1-7.3). The ⁸⁹Zr-labeled PAAm-DFO-DM1 conjugates were purified with a PD-10 column using PBS as the mobile phase. Radiochemical purity of the [⁸⁹Zr]Zr-PAAm-DFO-DM1 solution was assessed by instant thin layer chromatography (iTLC). Briefly, 5 µL of the reaction mixture was applied to an iTLC-SG chromatography paper (GE Healthcare) and developed using a 20 mM sodium citrate buffer at pH 5. The radioactivity on the paper was measured with a gamma detector. Each experiment was performed in triplicate. [⁸⁹Zr]Zr-PAAm-DFO-DM1 stability has been performed in FBS and in PBS with an excess of Ca²⁺ and Zn²⁺ compared to the ⁸⁹Zr. This allows to insure the stability also in terms of transmetallation. Each 25 µL of radiolabeled [⁸⁹Zr]Zr-PAAm-DFO-DM1 was diluted in 25 µL of either ZnCl₂ solution at 1 M pH 5 or CaCl₂ solution at 1 M pH 7 and incubated at 37 °C during 24h. Samples were analyzed similarly thanks to iTLC techniques. Each sample was repeated in triplicate.

## [⁸⁹Zr]Zr-PAAm-DFO-DM1 imaging and animal survival study

Mice were allocated to three experimental groups evaluating (i) the pharmacokinetics and biodistribution of PAAm with [⁸⁹Zr]Zr PET imaging comparing the intravenous injection to the subcutaneous injection, (ii) Tumor uptake derived from PET imaging correlating with antitumoral efficacy compared with an anti-CTLA4 immunotherapy on CT 26 model and (iii) on MC 38 model (Scheme S1). Ten mice (age 6 weeks; 18.9 ± 1.4 g) were used for the pharmacokinetic/biodistribution experiment.

For the first experimental experiment, intravenous administration (i.v., 1.9mg/160 µL, 4.06 ± 0.14 MBq; n = 5) was compared with subcutaneous (s.c., 1.9 mg/160 µL, 4.02 ± 0.12 MBq; n = 5) (Scheme S2). A 60-minute dynamic PET scan was performed immediately after injection of the [⁸⁹Zr]Zr-PAAm-DFO-DM1. 20-minute static PET scans were subsequently acquired at selected times (4, 24, 48, 72 h, 7 and 10 days).

In the antitumoral efficacy experiment, 36 Balb/c mice bearing CT26 tumors (age 6 weeks; 17.9 ± 1.2 g) were divided into four groups: (i) control, with intravenous injection of saline solution without PET imaging (125 µL, n = 6), (ii) 3.4 mg of [⁸⁹Zr]Zr-PAAm-DFO-DM1 intravenous injection (theoretically 6 mg of active ingredient/kg) at 6.32 ± 0.47 MBq/120 µL, n = 11, (iii) 1.97 mg of [⁸⁹Zr]Zr-PAAm-DFO-DM1 intravenous injection at 3.72 ± 0.26 MBq/120 µL, co-injected with 100 µg (5 mg/kg) anti-CTLA4/105 µL, n = 8 (iv) 100 µg (5 mg/kg) intravenous injection of anti-CTLA4/105 µL, n = 8.
same protocol was followed with 36 C57BI/6 mice bearing MC 38 tumors (age 9 weeks, 19.7 ± 0.6 g) with (i) Saline solution, n = 8, (ii) 4mg of [⁸⁹Zr]Zr-PAAm-DFO-DM1 at 6.38 ± 0.40 MBq/160 µL, n = 9, (iii) 4 mg of [⁸⁹Zr]Zr-PAAm-DFO-DM1 at 6.64 ± 0.33 MBq/120 µL with 100 µg anti-CTLA4/105 µL, n = 8 and (iv) 100 µg anti-CTLA4/105 µL, n = 8. A separate control group received injections solely of [⁸⁹Zr]-DFO to ascertain whether the distribution of the PAAm differed from that of unbound DFO, n = 3 bearing CT26 tumors. The mice were imaged at the time were the tumoral uptake reach its maximal accumulation determined in the first experimental group. 20-minute static PET scans were subsequently acquired at 72 h after injection. PET emission scans were performed using microPET scanners (Siemens). After treatment, tumors were measured twice a week with a caliper. Animal survival was determined in function of human endpoints. These endpoints were established considering criteria such as maximum acceptable tumor volume, weight loss in animals (not exceeding 20%), presence of surface necrosis posing infection risks, and overall animal health score.

For the three groups, PET images were reconstructed with the Inveon Acquisition Workspace software (v2.1) using the OP-OSEM3D-MAP algorithm. Normalization and corrections for dead-time, scatter, decay and attenuation were applied to all PET data. Volumes of interest (VOIs) were defined with the PMOD software (v4.4). Fixed- size spherical VOIs (1.25-3 mm³) were drawn in representative parts of the heart, liver, muscle, bone, gallbladder and kidneys. Mouse subcutaneous tumors were delineated semi-automatically. The mean activity concentration (kBq.cm⁻³) in each VOI was divided by the total injected dose (kBq) to obtain the percentage of injected dose per volume of tissue (%ID.cm⁻³).

## Pharmacokinetic modeling

Plasma activity concentrations were deduced from image-derived whole-blood activity concentrations from the left-heart ventricle. Plasma half-life was estimated by mono-exponential fitting thanks to GraphPad. As PAAm are restricted to the plasma, as determined by the separation of blood to plasma, a blood-to-plasma concentration ratio of 0.55 was used. The areas under the curve (AUC) of the i.v. groups were compared with those obtained from the s.c. group to obtain the bioavailable fraction (F) according to the following equation:

$$F = \frac{AUC_{sc} * dose_{iv}}{AUC_{iv} * dose_{sc}}$$

The bioavailable fraction refers to the proportion of a drug or other substance that enters the systemic circulation and is available to exert its biological effect.

## Statistics

Statistical analyses were performed with GraphPad Prism (v10.0.1). The influence of the route of administration and treatment was assessed by ANOVA followed by a pair-wise comparison of means with Bonferroni's *p*-value adjustment. When only the route of administration was considered, a two-tailed Student t-test was used. Results were considered statistically significant for *p*-values < 0.05.
# Results

## Radiosynthesis of the PAAm-based prodrug with $^{89}$Zr

DFO was incorporated into the PAAm scaffold through direct conjugation (see Figure SI1). 3% w/w of DFO was incorporated in the PAAm (all the NMR and SEC HPLC are described in supplementary information “Synthesis and Radiosynthesis of the $[^{89}Zr]Zr-PAAm-DFO-DM1$”). Radiolabeling was conducted using mild conditions subsequent to neutralizing $^{89}$Zr oxalate with a sodium carbonate solution. The radiopurity of $[^{89}Zr]Zr-PAAm-DFO-DM1$ was determined to be 95.6 ± 0.9% via iTLC analysis (Figures 1.A & 1.C). No additional purification steps were required prior to the injection of $[^{89}Zr]Zr-PAAm-DFO-DM1$. Notably, no radiolysis occurred in FBS even after a 24 h-incubation period (Figure 1.D). Similarly, incubation in PBS with excess $Ca^{2+}$ or $Zn^{2+}$ for 24 h revealed no signs of radiolysis or transmetallation.

A.

<figure><img src="image_4.png" /></figure>

B.

<figure><img src="image_5.png" /></figure>

C.

<figure><img src="image_6.png" /></figure>

**Figure 1.** Water-soluble PAAm-DFO-DM1 synthesis and radiolabeling. **A.** Representation of the PAAm-DM1 polymer prodrug containing 3% w/w DFO chelate moieties inserted in the PAAm chain (PAAm-DFO-DM1), followed by radiolabeling under mild condition (37°C, 1 h, pH = 7.2, HEPES) to yield $[^{89}Zr]Zr-PAAm-DFO-DM1$. **B.** Confirmation of the radiochemical purity by iTLC. **C.** Stability of the radiolabeled PAAm-DFO-DM1 in presence of calcium or zinc excess compared to vehicle (without any excess of calcium or zinc) to detect any transmetallation determined by iTLC during 24 h in FBS and.
Comparison between intravenous and subcutaneous injection of [⁸⁹Zr]Zr-PAAm-DFO-DM1

PAAm has previously shown promising results as a polymer promoiety for the delivery of paclitaxel via subcutaneous administration of water-soluble Ptx-PAAm prodrugs. Herein, we have demonstrated the potential of water-soluble PAAm-DM1 as a cytotoxic drug on tumor cells through an MTS assay (Figure SI2). Our aim was to compare i.v. vs s.c. injections to assess the versatility of the polymer prodrug across different administration routes. PET imaging of PAAm facilitated the determination of tissue distribution over time, notably within tumors. The polymer prodrug was administered to mice harboring two CT26 tumors. Notably, polymer prodrug exhibited a specific accumulation within the tumors compared to blood or muscle tissue regardless of the injection route. Ratios between tumor to blood or muscle are shown in Figure SI3. The tumor accumulation persisted up to 7 days post-injection. Tumor-to-muscle ratios seem to demonstrate a maximum accumulation of the [⁸⁹Zr]Zr-PAAm-DFO-DM1 72 h after injection. A rapid accumulation of PAAm within tumors was observed, reaching its peak 1 h after i.v. injection, whereas s.c. injection showed maximum accumulation at 24 h, as observed previously in local versus intravenous injection.¹⁸ However, the elimination profile and off-target accumulation of PAAm appeared similar regardless of the injection route (Figure 2). The only slight difference is in the liver, where accumulation is greater with i.v. injection than with s.c. injection. In the case of s.c. injection, the majority of the activity remained localized at the injection site, whereas i.v. injection resulted in a predominant renal elimination. The bladder shows the greatest difference between i.v. and s.c. injections; however, this does not accurately reflect the true distribution due to variability in the mice's urination and the timing of the imaging.
<figure><img src="image_7.png" /><figcaption>**Figure 2.** Kinetic tissue biodistribution analysis of [⁸⁹Zr]Zr-PAAm-DFO-DM1 via PET Imaging following intravenous or subcutaneous administration. **A. & C.** PET Images representative of i.v. and s.c. injections, highlighting: K. – Kidneys, L. – Liver, T. – Tumor, I. – Injection Site. **B. & D.** Distribution profiles extracted from PET Imaging analysis across various organs of interest over time.</figcaption></figure>
## Pharmacokinetics comparing blood and tumor accumulation

Based on our previous research on PAAm-Ptx polymer prodrugs and the confirmed stability of [⁸⁹Zr]Zr-PAAm-DFO-DM1, no metabolite formation is expected.⁷ The percentage of parent compound in plasma over time was well-described by a biexponential fit, with a terminal half-life of 34 ± 6 h (Figure 3). For s.c. injection, a single exponential fit described the percentage of the parent compound in plasma over time, considering the lack of earlier time point acquisition before 4 h post-injection, which precludes determining the contribution of rapid tissue distribution of the radioligand. The s.c. half-life was determined to be 23 ± 4 h, with no noticeable difference from the i.v. half-life. Drug exposure within the tumor during the 7-day post-injection period was quantified by the area under the curve. Notably, no significant differences were observed, whether considering the plasma or tumor compartments (Figure 3.C). The bioavailability of 94 % after s.c. injection confirmed that the fate of the polymer prodrug is similar between the two injection routes.

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<table><thead><tr><th></th><th>T<sub>1/2β</sub> (h.) plasma</th><th>AUC plasma (%ID.cm<sup>-3</sup>.h)</th><th>AUC Tumor (%ID.cm<sup>-3</sup>.h)</th></tr></thead><tbody><tr><td>i.v.</td><td>34 ± 6</td><td>177 ± 7</td><td>1820 ± 137</td></tr><tr><td>s.c.</td><td>23 ± 4</td><td>165 ± 11</td><td>1869 ± 218</td></tr></tbody></table>

**Figure 3. Pharmacokinetics of [⁸⁹Zr]Zr-PAAm-DFO-DM1 in plasma and in tumor.** Time activity curves in plasma (A.) and in tumors (B.) comparing i.v. and s.c. injections. C. Pharmacokinetic parameters (t₁/₂β and AUC in plasma and tumor) depending on the injection route. No significant difference has been observed comparing i.v. and s.c. injections.

i.v. administration was chosen for the therapeutic evaluation of PAAm-DM1 primarily to ensure precise control over drug plasma levels, which is essential for medications with a narrow therapeutic window, such as those used in PET imaging. It also reduces the risk of local irritation associated with repeated s.c. injections, improving patient comfort. Moreover, for patients needing continuous drug delivery, i.v. administration offers more consistent and stable dosing compared to s.c.

## Is the tumoral uptake reflecting the passive accumulation of the polymer prodrug?

We then examined the impact of the passive accumulation effect on [⁸⁹Zr]Zr-PAAm-DFO-DM1 distribution and its correlation with therapeutic efficacy. Combining immunotherapy with PAAm-DM1 was also explored to assess potential cumulative therapeutic efficacy. Prior to treatment, groups
were imaged with [⁸⁹Zr]Zr-PAAm-DFO-DM1 72 h after injection, coinciding with peak accumulation of the radiolabeled PAAm (Figure 3B). The distribution of [⁸⁹Zr]Zr-PAAm-DFO-DM1 was compared between the two groups of mice to validate the reproducibility of the EPR effect within the same model. Additionally, comparison with [⁸⁹Zr]Zr-DFO alone was conducted to ensure the specificity of radiolabeled PAAm accumulation. No significant difference in [⁸⁹Zr]Zr-PAAm-DFO-DM1 uptake was observed within the same model groups. However, a significant difference in uptake was noted between the two tumor models (MC38: tumor uptake = 2.89 ± 0.40 % ID.cm⁻³, CT26: tumor uptake = 1.25 ± 0.22 % ID.cm⁻³, p-value < 0.0001). Overall, the tumor model did not drastically influence distribution in other organs of interest, except for the liver. Low uptake of [⁸⁹Zr]Zr-DFO was observed in tumors (0.10 ± 0.02 % ID.cm⁻³).

<figure><img src="image_10.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_11.png" /></figure>
**Figure 4.** [⁸⁹Zr]Zr-PAAm-DFO-DM1 uptake in comparison with [⁸⁹Zr]Zr-DFO after intravenous injection. **A.** Representative PET images of [⁸⁹Zr]Zr-PAAm-DFO-DM1 before treatment with PAAm-DM1 alone or in combination with anti-CTLA4 in mice bearing CT26 tumors compared to [⁸⁹Zr]Zr-DFO distribution at 72 h post-injection. **B.** Biodistribution determined by PET imaging of [⁸⁹Zr]Zr-PAAm-DFO-DM1 in the two groups of treatment set in mice bearing CT26 or MC38 tumors compared to DFO distribution alone. highlighting: K. – Kidneys, L. – Liver, T. – Tumor, S. – Spleen.

**How accurately can we predict treatment efficacy solely through passive accumulation?**

In order to exploit the complementary mechanisms of action of chemotherapy and immunotherapy to enhance anti-tumor immune response, we administered [⁸⁹Zr]Zr-PAAm-DFO-DM1 to groups receiving PAAm-DM1 treatment alone or in combination with anti-CTLA4. The 6 mg.kg⁻¹ dose of PAAm-DM1 (DM1 equivalent) was selected based on the maximum tolerated dose (MTD) established during preliminary studies. The MTD for PAAm-DM1 was 10 mg.kg⁻¹ (DM1 equivalent), while free DM1 showed a much lower tolerance, with an MTD of only 0.5 mg.kg⁻¹ (Figure SI4). Therefore, free DM1 at a dose of 6 mg.kg⁻¹ was not included as a control due to its high toxicity. Previous studies have shown that a dose of 4 mg.kg⁻¹ of free DM1 results in a survival time of no more than 6 days in healthy mice.¹⁹ Following discussions with the ethics committee, it was deemed unethical to administer a higher dose, as the severe suffering and rapid mortality would outweigh any potential scientific value. Tumor growth was monitored until reaching predefined human endpoints for all four treatment groups. While PAAm-DM1 stabilized tumor growth, anti-CTLA4 notably reduced tumor volume (Figure 5.A&B). Notably, no significant differences were observed in treatment efficacy between the CT26 model groups, although anti-CTLA4 demonstrated superior efficacy in MC38. Combination therapy with anti-CTLA4 and PAAm-DM1 did not significantly enhance efficacy compared to anti-CTLA4 alone. Given these findings, we sought to explore the correlation between the EPR effect, as indicated by maximal tumor uptake of [⁸⁹Zr]Zr-PAAm-DFO-DM1, and tumor growth. Surprisingly, no correlation was found between [⁸⁹Zr]Zr-PAAm-DFO-DM1 uptake and treatment efficacy across all treatment groups (Figure 5.C – 5.F). Furthermore, comparing tumor growth and PAAm-DM1 accumulation within the tumor between MC38 and CT26 models revealed no discernible correlation (Figure 5.G). We did not observe any toxicity based on the behavioral and clinical signs (body weight, body score from Navarro et al.²⁰ and also feeding behavior and neurological symptoms).
A.

<figure><img src="image_12.png" /></figure>

B.

<figure><img src="image_13.png" /></figure>

C.

PAAm-DFO-DM1

<figure><img src="image_14.png" /></figure>

D.

PAAm-DFO-DM1+anti-CTLA4

<figure><img src="image_15.png" /></figure>

E.

PAAm-DFO-DM1

<figure><img src="image_16.png" /></figure>

F.

PAAm-DFO-DM1+anti-CTLA4

<figure><img src="image_17.png" /></figure>

G.

All groups

<figure><img src="image_18.png" /></figure>

**Figure 5.** Treatment efficacy correlated with tumor uptake. A. Tumor growth in CT26 model with different treatments (PAAm-DM1 alone, combined with anti-CTLA4, or anti-CTLA4 alone, all compared with the control group with vehicle only (Sham)). Vehicle group is strictly significant compared to the other treatment groups (*p*-value < 0.0001 % ****). B. Tumor growth in MC38 model in the different treatment groups. Note that not only the vehicle is significantly different from all the treatments (*p*-value < 0.0001 % ****) but also the tumor growth from the anti-CTLA4 group is significantly lower than the other treatments (*p*-value < 0.01 %). Correlation between tumor growth and tumor uptake of [⁸⁹Zr]Zr-PAAm-DFO-DM1 at 72 h post-injection in CT26 model with PAAm-DM1 treatment (C.), combined with anti-CTLA4 (D.), in MC38 model with PAAm-DM1 treatment (E.), or combined with anti-CTLA4 (F.). All the tumor uptakes were finally compared with their respective [⁸⁹Zr]Zr-PAAm-DFO-DM1 tumor uptake at 72 h post injections (G.).
Discussion

Addressing the shortcomings of conventional chemotherapy, such as lack of tumor-targeting specificity and treatment resistance, is crucial, and our polymer prodrug strategy offers a promising solution by enhancing passive accumulation in tumor tissues. We turned to the remarkable macromolecular structure of PAAm-based prodrug, as its ability as a carrier for therapeutic agents, particularly chemotherapeutic drugs, makes it a potent lever for improving patient care.⁷ Investigation of PAAm-based prodrug for s.c. administration aims particularly to rationalize injection times, potentially paving the way for patient self-administration at home—an approach markedly distinct from i.v. administration. S.c. administration significantly abbreviates injection durations for example, as demonstrated by our team, showcasing the manifold benefits of this strategy. At first, our focus lies in pharmacokinetic comparison between these two administration routes, leveraged by the most quantitative, whole-body, and dynamic biomedical imaging techniques available, PET imaging. The radiolabeling of polymer prodrugs for assessing their tissue distribution is a pivotal aspect in predicting their efficacy. Historically, much of the PET imaging strategy has been based on indirect imaging of polymer prodrug efficacy, often through targeting predictive biomarkers.²¹,²² However, it is essential to recognize that the pharmacokinetics of the polymer prodrug itself can serve as a predictive indicator. Therefore, the development of a theranostic platform incorporating PET imaging holds great potential for improving drug/prodrug evaluation and personalized treatment approaches.²³⁻²⁵ The radiolabeling of PAAm proved to be remarkably uncomplicated. Intriguingly, the administration of the radiolabeled PAAm conjugated to DM1 exhibited consistent tumor exposure, irrespective of the injection route employed. The delay of diffusion that lead to a delay of tumor accumulation between local injection and direct i.v. injection is explained by the diffusion from the injection site to blood compartment.¹⁸ However, we noted substantial bioavailability, reflecting efficient absorption and abundant availability of the drug for therapeutic endeavors. This aligns with the conclusions drawn by Klose et al., who compared administration routes of various radiolabeled peptides, reaching a similar consensus.²⁶ Their investigation into the biodistribution of theranostic ligands post-s.c. or intraperitoneal application demonstrated nearly equivalent outcomes to those following i.v. injection. We observed not difference in terms of toxicity based on the physiologic observation of the mice between the two injection routes. PET imaging confirmed the predominant elimination route of PAAm-DM1, primarily through renal excretion followed by hepatic clearance, in accordance with lowering the toxicity of DM1 thanks to its conjugation with high hydrophilic prodrug.

DM1 has been extensively combined with antibody to increase tumor specificity and minimalizing the off-target toxicity. This antibody-drug conjugate strategy presents mixed results in tumor efficacy and is highly depend on biomarker.²⁷ Compared to ADCs, our polymer prodrug has the advantage of targeting a broader range of tumor types without relying on protein expression. Regardless of the injection route, it demonstrates significantly lower liver uptake than ADCs, along with a high rate of
renal elimination, potentially resulting in improved long-term toxicity profiles. Our study explored a different strategy, but further direct comparisons with ADCs are required to assess its relative benefits. Ran et al. have pioneered the development of a hydrophilic peptide named SAMPD, as an alternative strategy, enhancing DM1's biocompatibility while preserving its tumor efficacy.²⁸ Their research demonstrated a safe DM1 release with minimal toxicity. However, it also showed high liver uptake due to the nanostructured DM1 polymer prodrug assembly, which could potentially lead to long-term toxicity.

Our investigation also focused on combination therapy exploration with particularly immunotherapy targeting the immune checkpoint CTLA4, which has shown interesting results in the CT26 and MC38 colon models.¹⁸ This combination, called chemoimmunotherapy, represents a promising frontier in cancer treatment, offering significant advancements in patient care by synergizing chemotherapy with immunotherapy.²⁹ Combining anti-CTLA4 with PAAm conjugated to DM1 holds considerable potential for markedly reducing tumor growth. This combination strategy capitalizes on immunotherapy's ability to combat disseminated and metastatic cancer, which contrasts with its relative inefficacy against solid tumor masses.³⁰ While we successfully showcased the tumor-reducing potential of PAAm-DM1 alone, the effectiveness of the combination therapy was somewhat subdued. In the immunogenic CT26 model, anti-CTLA4 therapy exhibited near-complete efficacy, leaving little room for improvement. However, in the less immunogenic MC38 model, we expected complete tumor remission with the combination therapy, which was not observed. Parra et al., in their exploration of metronomic chemotherapy combined with anti-CTLA4, arrived at similar conclusions in more resistant tumor models.³¹ Enhancements in the optimal dosing, timing, and sequencing of these treatments present avenues for improvement, although our dosage and treatment sequence were based on our previous studies.

Following these observations, PET imaging of PAAm-DM1 revealed that the passive accumulation of PAAm did not correlate with drug efficacy across different treatment arms. We had expected observing a relationship between polymer prodrug tumor accumulation by passive accumulation and tumor outcomes. PET imaging should have been a reliable biomedical technic to overcome the heterogeneity of the passive accumulation observed in patient.⁸,³²⁻³⁵ Several factors may account for this discrepancy. Firstly, there appears to be a lack of passive accumulation heterogeneity in the tumor within the same model. In our study, we incorporated two distinct tumor models, each exhibiting unique EPR effects which can lead to heterogeneous passive accumulation, with the expectation of observing divergent efficacy outcomes for PAAm-DM1. To our surprise, this anticipated variation did not manifest. It appears that the predictive capacity of the passive accumulation alone regarding PAAm-DM1 efficacy is insufficient, likely due to the complex interplay between the polymer prodrug and the tumor microenvironment. The immune system can be a crucial factor in drug distribution. For example, it has been shown that the immune system can alter the pharmacokinetics of chemotherapy
by influencing drug metabolism and clearance. Activated macrophages may sequester or metabolize
these drugs, affecting their availability and efficacy.³⁶ This highlights the challenge of predicting
efficacy based solely on imaging and suggests the need for further investigation. Prior discussions
have highlighted the reciprocal influence between chemotherapy efficacy and the immune
response.³⁷,³⁸ Furthermore, the heterogeneity of drug release adds complexity, and it would be valuable
to visualize PAAm delivery alongside DM1 delivery. However, this presents a challenge as accurately
quantifying DM1 without altering its structure while dynamically analyzing its distribution remains
technically demanding.

Conclusion

In conclusion, our study marks the pioneering use of PET imaging for visualizing PAAm-based
polymer prodrug in chemotherapy delivery, particularly when conjugated to DM1. Regardless of
whether administered via s.c. or i.v. injection, both routes resulted in equivalent distribution,
culminating in comparable tumor exposure to the drug. Leveraging the EPR effect, PAAm exhibited a
heightened specificity towards tumor tissues. While PAAm-DM1 showcased significant efficacy, the
link between efficacy and tumor uptake mediated by the EPR effect remains unestablished and
warrants further biological investigation. Such endeavors are crucial for unraveling the influence of
the tumor microenvironment on PAAm-DM1 efficacy, consequently shedding light on the limited
success observed in combining PAAm-DM1 with immunotherapy to enhance patient outcomes.

Acknowledgements

This work was performed on an imaging platform member of the France Life Imaging
network (grant ANR-11-INBS-0006). The authors acknowledge the financial support from
Agence Nationale de la Recherche - ANR (ANR-20-CE17-0038, ELUSUB).

Abbreviations

ADCs = Antibody Drug Conjugates, API = Active Pharmaceutical Ingredients, CTLA4 = Anti-Cytotoxic T-Lymphocyte-Associated protein 4, DM1 = Mertansine, EPR = Enhanced Permeability Retention, FBS = Fetal Bovine Serum, iTLC = instant Thin Layer Chromatography, NMR = Nuclear Magnetic Resonance, PAAm = Polyacrylamide, PBS = Phosphate-Buffered Saline, PET = Positron Emission Tomography, Ptx = Paclitaxel, VOI = Volumes Of Interest.
# Declaration of Generative AI and AI-assisted technologies in the writing process

No AI and AI-assisted technologies have been used on the paper.

## Declaration of interest

AB, TB, NT and JN are co-founders of the startup company Imescia.

## Author contributions

Conceptualization: JN, NTsapis, NTournier, TB, CT; Data curation: SMA, KA, VLT, AB, NB, DK, LP; Formal analysis: SMA, KA, AB, TB, CT; Funding acquisition: NTsapis, NTournier, TB, CT; Investigation: SMA, KA, VLT, AB, NTsapis, NTournier, JN, TB, CT; Methodology: TB, CT; Project administration: NTournier, TB, CT; Resources: NTsapis, JN, TB, NTournier, CT; Software: NTournier, CT; Supervision: TB, CT; Validation: NTsapis, NTournier, JN, TB, AB, CT; Roles/Writing - original draft: SMA, TB, CT; and Writing - review & editing: SMA, NTsapis, NTournier, JN, TB, AB, CT

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