# Antibody-Vincristine Conjugates as Potent Anticancer Therapeutic Agents

Agathe Boos, Julien Most, Héloïse Cahuzac, Louis Moreira da Silva, François Daubeuf, Stéphane Erb, Sarah Cianférani, Oscar Hernandez-Alba, Constantin Semenchenko, Igor Dovgan, et al.

► To cite this version:

Agathe Boos, Julien Most, Héloïse Cahuzac, Louis Moreira da Silva, François Daubeuf, et al. Antibody-Vincristine Conjugates as Potent Anticancer Therapeutic Agents. Journal of Medicinal Chemistry, 2024, 10.1021/acs.jmedchem.4c02425. [DOI: 10.1021/acs.jmedchem.4c02425](https://doi.org/10.1021/acs.jmedchem.4c02425)

HAL Id: hal-04856907

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Submitted on 27 Dec 2024

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# Antibody-Vincristine Conjugates as Potent Anti-Cancer Therapeutic Agents

Agathe Boos,ª Julien Most,ᵇ Héloïse Cahuzac,ᵇ Louis Moreira da Silva,ᵇ François Daubeuf,ᶜ Stéphane Erb,ᵈ Sarah Cianférani,ᵈ Oscar Hernandez-Alba,ᵈ Constantin Semchenko,ᵉ Igor Dovgan,ᵉ Sergii Kolodych,ᵉ Alexandre Detappe,ᵉ Françoise Dantzer,ª Alain Wagner,ᵇ Maria Zeniou *ª and Guilhem Chaubet *ᵇ

**Abstract.** Antibody drug-conjugates (ADC) are a well-established class of therapeutics primarily used in oncology to selectively deliver highly cytotoxic agents into cancer cells. While ADC should theoretically spare healthy tissues and diminish side effects in patients, off-target toxicity is still observed, all the more serious as the drugs are extremely potent. In the quest towards safer payloads, we used the conventional chemotherapeutic drug vincristine to develop antibody-vincristine conjugates. Vincristine was N-alkylated with a cleavable linker and the resulting linker-payload conjugated to free cysteines of antibodies. We show that trastuzumab-vincristine conjugates display subnanomolar potency in vitro on HER2-positive cells, two orders of magnitude lower than free vincristine and comparable with marketed ADC. In vivo, trastuzumab-vincristine conjugates led to remarkable efficacy when compared to two standards of care, with complete tumor regression just nine days after single administration. This highlights the untapped potential of the chemotherapeutic arsenal towards the development of novel ADC.

## Introduction

The successful development of antibody-drug conjugates (ADC) in the early 2000's was the fruit of a decades-old dream: to selectively deliver cytotoxic drugs into cancer cells without damaging healthy cells.¹,² To compensate for the minute amounts of ADC reaching their target (~1% of the injected dose),³ it was envisioned that highly cytotoxic drugs were needed. With potency in the 1-10 picomolar range, making them unsuitable for systemic delivery, DNA-binding agents such as calicheamicins and microtubule polymerization inhibitors including auristatins or maytansinoids, were hence employed in cancer therapy.⁴,⁵ However, despite a convincing development rationale, undesired side toxicity is still observed with ADC, whether due to premature payload deconjugation leading to off-target off-tumour cytotoxicity, or to their on-target off-tumour internalization in healthy antigen-expressing cells.⁶⁻⁹ Consequently, almost all ADC are riddled with severe adverse effects, such as neutropenia, thrombocytopenia and hepato- or cardiotoxicity.⁶,¹⁰⁻¹²

Among other developments in the field of ADC, recent advances in linker design allowed the conjugation of higher amounts of less toxic payloads, such as some topoisomerase I inhibitors.¹³ The addition of more hydrophilic chemical functions such as polyethylene glycol (PEG) groups or sulfonate moieties helped to counterbalance the inherent hydrophobicity of the payloads

and produce ADC with higher drug-to-antibody ratios (DAR) and lower risks of aggregation.¹⁴,¹⁵ This was paralleled by the addition of novel drugs to the ADC' toolbox that are less hydrophobic than members of auristatin, maytansine, and calicheamicin families, leading to ADC with improved stability and pharmacokinetics but also with lower immunogenicity.¹⁶⁻¹⁸ Additionally, focusing on the conjugation of thiol groups, borne by the side-chains of the eight solvent-accessible cysteine residues involved in the antibody's interchain disulfide bonds, has resulted in higher and more homogeneous DAR.¹⁹ Recently, two ADC with high DAR (~ 8) – sacituzumab govitecan (marketed as Trodelvy®), an anti-TROP2 antibody conjugated to the topoisomerase I inhibitor SN38 and indicated in the treatment of triple-negative breast cancer, and trastuzumab deruxtecan (Enhertu®), bearing the topoisomerase I inhibitor Dxd and prescribed against HER2-positive (HER2+) cancers – have reached the clinic.[17,18] Most importantly, although the potency of the free forms of SN38 and Dxd is lower than that of the payloads used in the previous generations of ADCs (typically in the low picomolar to high nanomolar range), both Trodelvy® and Enhertu® still contributed significantly to patient survival (Figure 1).²⁰⁻²³

<footnote>ª Poly(ADP-ribosylation and Genome Integrity, Laboratoire d'Excellence Medalis, UMR7242, Centre National de la Recherche Scientifique/Université de Strasbourg, Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg, 300 bld. S. Brant, CS10413, 67412 Illkirch, France; Strasbourg Drug Discovery and Development Institute (IMS), Strasbourg, 67000, France</footnote>

<footnote>ᵇ Bio-Functional Chemistry, Laboratoire d'Excellence Medalis, UMR7199, Centre National de la Recherche Scientifique/Université de Strasbourg, Faculté de Pharmacie de Strasbourg, 74 Route du Rhin, 67400 Illkirch-Graffenstaden, France; Strasbourg Drug Discovery and Development Institute (IMS), Strasbourg, 67000, France.</footnote>

<footnote>ᶜ PCBIS (CNRS - UAR 3286), ESBS - Pôle API, 300, boulevard Sébastien Brant, CS 10413, 67412 Illkirch-Graffenstaden Cedex, France</footnote>

<footnote>ᵈ BioOrganic Mass Spectrometry, Laboratoire d'Excellence Medalis, UMR7178, Centre National de la Recherche Scientifique/Université de Strasbourg, Institut pluridisciplinaire Hubert Curien, 23 rue du Lœss, BP 28, 67037 Strasbourg Cedex 2, France; Infrastructure Nationale de Protéomique ProFI - FR2048, 67087 Strasbourg, France; Strasbourg Drug Discovery and Development Institute (IMS), Strasbourg, 67000, France</footnote>

<footnote>ᵉ Syndivia SAS, Syndivia, 8 allée Gaspard Monge, Strasbourg 67083, France</footnote>

<footnote>ᵉ Carbogen Amcis AG, Hauptstrasse 171, 4416 Bubendorf, Switzerland</footnote>

<footnote n="8">⁸ Institut de Cancérologie Strasbourg Europe, Strasbourg, France; Équipe labellisée Ligue contre le Cancer; Strasbourg Drug Discovery and Development Institute (IMS), Strasbourg, 67000, Franc</footnote>

<footnote>† These authors contributed equally to the work.</footnote>

<footnote>* Corresponding authors: zeniou@unistra.fr; chaubet@unistra.fr</footnote>
The successful entry on the market of these two ADC demonstrates the interest in exploring less toxic but more hydrophilic payloads than those of the previous generations, in order to improve the therapeutic index of ADC. In this quest, we turned our attention toward famous antimicrotubule agents: vinca alkaloids. With a toxicity 10-to-1000 times lower, coupled with a less pronounced hydrophobic character due to the presence of protonable tertiary amines (see Supporting Information),²⁴,²⁵ we assumed that they could compete with the two reference payloads in this class of drugs classically employed in a wide range of ADC, namely maytansinoid DM1 and monomethyl auristatin E (MMAE) (Figure 1).²⁶,²⁷

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Families, names, and chemical structures of a selection of anticancer drugs with documented or hypothetical applications in ADC. The approximate cytotoxicity ranges of the drugs are given as IC₅₀ values and come from various sources of the literature (dataset GDSC2 from release 8.5 (October 2023) of the cancerRxgene platform was used).²⁸⁻³¹</figcaption></figure>

While several members of the vinca alkaloid family, such as vinblastine and vincristine, were approved in the 1960's as chemotherapeutics for the treatment of several types of cancer,²⁸ vinca alkaloid-based conjugates have only been scarcely investigated.²⁹ One of the only mention in the literature is that of an antibody-vinblastine conjugate, described in the late 1980s, but whose clinical development was halted due to poor efficacy during early trials.³⁰ Considering the tremendous developments seen in the field of ADC over the past decades, we sought to investigate vinca alkaloids once again under the light of improved conjugation techniques and linker design.

## Results and Discussion

In order to do so, we adapted a bioconjugation method of tertiary amine-containing drugs, described in 2016 by scientists at Genentech (Figure 2).³¹ In this report, they demonstrated that several drugs could be regioselectively alkylated with a linker composed of a maleimidocaproyl (mc) valine-citrulline

(Val-Cit) motif equipped with an electrophilic *p*-aminobenzyl chloride (PAB-Cl) handle. The use of the Val-Cit dipeptide unit allows the cathepsin-mediated cleavage of the linker in the lysosomal compartment of targeted cells,³² while the presence of the self-immolative PAB spacer ensures the rapid and traceless release of the drug.³³ In addition, the mc group reacts selectively with thiols, allowing conjugation of the linker on cysteine residues through *thio*-Michael addition.³⁴ While maleimide are notoriously prone to *retro*-Michael addition, causing linker-payload deconjugation, this instability might nonetheless contribute to the antitumor effect of ADC, explaining why this linker still equips most of ADCs on the market and in clinical development.³⁵,³⁶

Using vinblastine as payload in the presence of tetrabutylammonium iodide (TBAI), they managed to isolate the corresponding ammonium – albeit in a poor 18% yield – but did not engage in further conjugation studies with this system. Selecting vincristine **1** as our model vinca alkaloid, we thus evaluated its alkylation with mc-Val-Cit-PAB-Cl **2** (Figure 2). However, in our hands, disappointing results were obtained following literature procedure,³¹ urging us to optimize this reaction further. Suspecting that N,N-diisopropylethylamine (DIPEA) might compete with **1** for alkylation, we turned our attention toward inorganic bases and found out that potassium carbonate (3.0 equiv.) in DMF could successfully deliver the expected ammonium adduct with improved yields (60% after 72 h at 25 °C). In an attempt to accelerate this conversion, a small volume of water (3% v/v) was added to the reaction mixture, in

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Preparation of the linker-drug payload. The literature procedure is that of Staben and coworkers.³⁵</figcaption></figure>

order to increase the solubility of the base and the reaction was performed at 37 °C for 24 h. Under these conditions, target compound **3** was isolated with an improved yield of 74% after purification (Figure 2). As expected, in-depth structural investigations by NMR spectroscopy indubitably confirmed the regioselectivity of the alkylation, identifying the tertiary amine of the catharanthine subunit as the sole modification site (see Supporting Information).
We next evaluated the conjugation of two therapeutic monoclonal antibodies (mAb) with our linker-payload: the anti-HER2 trastuzumab 4, used in the treatment of HER2-positive breast cancer and B-cell, non-Hodgkin's lymphomas, and rituximab 5, an anti-CD20 mAb used for the treatment non-Hodgkin lymphoma and certain leukaemia. To obtain ADC with average (av.) DAR equals to either 4 or 8, the interchain disulfide bonds of both antibodies were either partially or totally reduced using 2 or 20 equivalents of the reducing agent tris(2-

carboxyethyl)phosphine (TCEP), respectively, at pH 6.5 in phosphate-buffered saline (PBS) solution (Figure 3A). The conjugation reactions were performed in the same solvent, for 2 h at 25 °C before the resulting conjugates were analyzed by native mass spectrometry (nMS). These analyses revealed an excellent level of conjugation of the linker-payload 3 to both model mAb. As anticipated, partial reduction of interchain disulfide bonds led to an average DAR of 3.6 for trastuzumab-vincristine (T-VCR) 6a with only 3% of unconjugated (D0) mAb

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Production, characterization and in vitro cytotoxicity assays of trastuzumab- and rituximab-vincristine conjugates. **A:** influence of the amount of reducing reagent on the average (av.) DAR of the final trastuzumab-vincristine (T-VCR) and rituximab-vincristine (R-VCR) conjugates. **B-E:** SEC chromatograms and deconvoluted native mass spectra of T-VCR 6a (av. DAR = 3.6; B), T-VCR 6b (av. DAR = 7.8; C), R-VCR 7a (av. DAR = 3.6; D), and R-VCR 7b (av. DAR = 7.4; E) – red and yellow asterisks indicate linker-payload fragmentation (- 821 Da) and substitution of vincristine by a phosphate group (- 728 Da), respectively (see SI). **F-H:** cells were seeded at an appropriated density in a 96-well plate and allowed to adhere for 24 h prior to treatment with the indicated concentrations of each compound. Cell viability was measured 72 h later using the WST-1 cell proliferation assay. Data are represented as mean values ± SEM from at least three independent experiments (n = 3) and are expressed as the percentage of living cells relatively to untreated SKBR3 (F), and MDA-MB-231 cells (G). The table (H) recapitulates IC₅₀ values ± SEM of vincristine 1 and T-VCR 6b for cell lines included in this study. Curve-fits and IC₅₀ values were obtained using the GraphPad Prism software. NA: not applicable.</figcaption></figure>
left, while full reduction conditions led to homogeneous conjugates **6b** (average DAR 7.8), with only trace amounts of D6 being detected (Figure 3B and 3C).

Similar results were obtained for rituximab-vincristine (R-VCR) **7** conjugates (Figure 3D and 3E), confirming the versatile applicability of our approach to other mAb. In vitro cytotoxicity of newly synthesized **T-VCR 6a** and **6b** was next evaluated in several cancer cell lines overexpressing HER2 (HER2+) – i.e., SKBR3 and BT474 breast carcinoma cells, and NCI-N87 gastric carcinoma cells. The triple negative MDA-MB-231 breast cancer cell line, expressing very low levels of the HER2 surface antigen (HER2<sup>low</sup>), was used as a negative control cell line in these experiments. **R-VCR 7a** and **7b** were used as negative control ADC since all the cell lines used in these assays express very low levels of CD20.

As shown in Figures 3F and 3G, both SKBR3 and MDA-MB-231 displayed sensitivity to free vincristine **1** in the 10<sup>-8</sup>–10<sup>-9</sup> M range (similar results were obtained with BT474 and NCI-N87; see Supporting Information). Excitingly, **T-VCR 6b** exhibited

significantly higher cytotoxicity compared to free vincristine in all three HER2+ cell lines tested (i.e., SKBR3 cells: IC<sub>50</sub> (VCR) = 1.1 ± 0.3 nM vs IC<sub>50</sub> (T-VCR **6b**) = 0.031 ± 0.008 nM, ~30- to 40-fold increase; NCI-N87 cells: IC<sub>50</sub> (VCR) = 2.3 ± 0.5 nM vs IC<sub>50</sub> (T-VCR **6b**) = 0.27 ± 0.15 nM, ~50-fold increase; BT474 cells: IC<sub>50</sub> (VCR) = 0.3 ± 0.1 nM vs IC<sub>50</sub> (T-VCR **6b**) = 0.025 ± 0.006 nM, ~10-fold increase; see Figure 3H and Supporting Information). Similar gains in potency were measured for **T-VCR 6a**, with IC<sub>50</sub> values in the 10<sup>-10</sup>–10<sup>-11</sup> M range, two orders of magnitude lower than free vincristine (see Supporting Information) and in the range of currently marketed ADC. This could be explained by the presence of a basic, tertiary amine on both catharanthine and vindoline subunits of vincristine (pK<sub>a</sub> = ~ 7.5 and pK<sub>a</sub> ~5.5, respectively),<sup>37</sup> making the free drug positively charged at physiological pH and thus less prone to diffuse through cell membranes. More importantly, HER2-selectivity of the conjugates and the need for an active delivery of vincristine into cells to induce toxicity was demonstrated by an absence of toxicity of **T-VCR** on the HER2<sup>low</sup> cell line MDA-MB-231 at tested

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

<table><thead><tr><th rowspan="2">cells</th><th rowspan="2"></th><th colspan="3">IC<sub>50</sub> (nM)</th></tr><tr><th>VCR 1</th><th>T-VCR 6b</th><th>T-MMAE 8b</th><th>T-DM1</th></tr></thead><tbody><tr><td></td><td>SKBR3</td><td>1.0 ± 0.1</td><td>0.02 ± 0.01</td><td>0.15 ± 0.05</td><td>0.066 ± 0.016</td></tr><tr><td></td><td>MDA-MB-231</td><td>1.6 ± 0.8</td><td>NA</td><td>NA</td><td>NA</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Figure 4.** In vitro cytotoxicity assays of T-VCR conjugate **6b** in comparison with standard trastuzumab-based ADC T-MMAE and T-DM1. A-B: cells were seeded at an appropriated density in a 96-well plate and allowed to adhere for 24 h prior to treatment with the indicated concentrations of each compound. Cell viability was measured 72 h later using the WST-1 cell proliferation assay. Graphs represent mean values ± SEM from at least three independent experiments (n = 3) and are expressed as the percentage of living cells relatively to untreated SKBR3 (A) and MDA-MB-231 cells (B). C: table recapitulating IC<sub>50</sub> values ± SEM of VCR **1**, T-VCR **6b**, T-MMAE **8b** and T-DM1 for cell lines included in this study. Curves and IC<sub>50</sub> values were obtained using the GraphPad Prism software. NA: not applicable. D: chemical structures of T-MMAE and T-DM1 conjugates.</figcaption></figure>
doses (see Figure 3G and Supporting Information). This was further confirmed by the fact that under the experimental conditions used in this study (compound concentration), neither of the two **R-VCR** conjugates **7a** and **7b** nor unconjugated trastuzumab **4** were able to significantly affect the viability of HER2⁺ and HER2<sup>low</sup> cell lines (see Figures 3F and 3G, and Supplementary Information). This selectivity profile is consistent with the high stability of the conjugates in biological environments, as assessed in human and murine plasma (see Supporting Information). Considering the high potency of our **T-VCR** conjugates, we decided to compare them against both KADCyla®, a trastuzumab-DM1 (**T-DM1**) conjugate with an average DAR of 3.5 and standard of care in HER2⁺ cancers, and a trastuzumab-MMAE (**T-MMAE**) conjugate, with an average DAR of either ~2 (**T-MMAE 8a**) or ~8 (**T-MMAE 8b**) (Figure 4). In SKBR3 cells, similar to slightly superior cytotoxic effects of **T-VCR** (IC₅₀ 6a = 0.028 ± 0.016 nM; IC₅₀ 6b = 0.02 ± 0.01 nM) was observed compared with **T-DM1** (IC₅₀ = 0.066 ± 0.016 nM) and **T-MMAE** (IC₅₀ 8a = 0.056 ± 0.016 nM; IC₅₀ 8b = 0.15 ± 0.05 nM; see Figure 4A and Supplementary Information). In the latter case, the level of cytotoxicity observed was comparable to that of the free drug (IC₅₀ MMAE = 0.060 ± 0.002 nM), in good agreement with the literature,¹⁸,³⁸,³⁹ while the less pronounced

toxicity of **8b** could be due to aggregation issues.⁴⁰ As expected, no toxicity was observed in HER2<sup>low</sup> MDA-MB-231 cells for either of these conjugates, irrespective of their average DAR (Figure 4B and Supplementary Information).

To conclude these in vitro studies, we finally evaluated the bystander effect of vincristine. Bystander effect is an important characteristic of cytotoxic agents, reflecting their ability to passively cross membranes and diffuse into neighbouring cells after being released in target cells. A high bystander effect is beneficial for targeting heterogeneous tumours, but can cause toxicity to healthy tissues located near tumour cells. Therefore, it is crucial to find the right balance of the bystander effect based on target expression and tumour location. To evaluate this effect of vincristine-based conjugates, we prepared a conjugate of vincristine with a PSMA-targeting antibody, J591, with an average DAR of 4.5 (J591-VCR). Using the same antibody, we also prepared a benchmark ADC with a high bystander effect (J591-MMAE) and a benchmark ADC with a low bystander effect (J591-MMAF). The bystander effect was measured by co-seeding PSMA-positive LNCaP and PSMA-negative, EGFP-expressing SKBR3 EGFP cells, treating them with J591 conjugates and measuring the proportion of LNCaP and SKBR3 cells by flow cytometry. All J591 conjugates exhibited

<figure><img src="image_7.png" /><figcaption>**Figure 5.** In vivo cytotoxicity assays of T-VCR conjugate 6b in comparison with standard trastuzumab-based ADC T-DM1 and T-Dxd. **A-C:** NOD-SCID mice with subcutaneous heterotopic SKBR3 tumor cell xenografts were subjected to a unique intravenous injection of either PBS 1X (vehicle; grey) or 5 mg.kg⁻¹ of T-VCR 6b (green), **T-Dxd** (Enhertu®; yellow) **10**, or **T-DM1** (Kadcyla®; purple) **9**. **A:** tumor volume was measured every 2-3 days for 60 days. Experimental results are expressed as means of the percentage of remaining tumor volume +/- SEM (n = 5 for **T-DM1**, n = 6 for all other groups). **B:** Percentage of mice without complete tumor regression post-injection is represented as a function of time. Statistical analysis of differences between groups was performed using a Log-rank (Mantel-Cox) test with GraphPad Prism 7.0. Statistically significant differences were observed between T-VCR 6b and T-Dxd **10** (p value 0.0235 = < 0.05 (*/)) and T-VCR 6b and T-DM1 **9** (p value 0.0087 = < 0.01 (**)). **C:** percentage of surviving mice post-injection is represented as a function of time. Statistically significant differences were observed between groups (Log-rank (Mantel-Cox) test, p value 0.0165 = < 0.05 (*/)). **D:** chemical structure of **T-Dxd** conjugate.</figcaption></figure>
potent cytotoxic activity on LNCaP cells ($IC_{50}$ J591-VCR = 0.31 nM; $IC_{50}$ J591-MMAE = 0.32 nM; $IC_{50}$ J591-MMAF = 0.38 nM) and no activity on SKBR3 EGFP cells (i.e., $IC_{50}$ > 10⁻⁸ M; see Supplementary Information). Interestingly, J591-VCR displayed a pronounced bystander effect compared with the negative control J591-MMAF (i.e., 95% versus no bystander effect, respectively), but lower than that of J591-MMAE (99%), demonstrating the potential for a better balance between efficacy in heterogeneous tumours and toxicity in non-target tissues (see Supplementary Information).

Having proven that vincristine conjugates were both potent and selective in vitro, we envisioned that they could represent an attractive option toward the development of safer ADC. Indeed, based on our previous results, similar on-tumour efficacy between T-VCR, T-DM1, and T-MMAE but diminished off-target toxicity in the case of vincristine conjugates were expected in vivo – free vincristine being way less potent than the two other drugs, premature deconjugation of the payload should be less damageable.

To validate our assumption, the activity of our T-VCR conjugate 6b was assayed in immunodeficient NOD-SCID mice xenografted subcutaneously with SKBR3 cells. The animals were bred for eight weeks until sufficient tumour growth occurred before being split into four groups of five to six mice each. Each animal of a given group received a single intravenous injection of either the vehicle (PBS 1X, negative control) or one of three following ADC, all at the same concentration of 5 mg.kg⁻¹: T-VCR 6b, T-DM1 9 (Kadcyla®, positive control), and T-Dxd 10 (Enhertu®, positive control). Tumour volume measurements revealed a remarkable antitumor activity of our T-VCR conjugate, with complete tumour regression observed only nine days after injection, whereas complete tumour regression was only observed at later time points (> 20 days) in mice treated with any of the two positive control ADC (Figure 5A and B). Moreover, no apparent toxicity was observed up to 60 days post-injection in mice receiving T-VCR 6b, as determined by bodyweight loss, and neither tumour relapse nor metastasis, as shown by the survival curves in Figure 5C (see also Supporting Information). Conversely, toxicity was observed in half the mice of the T-Dxd group, materialized by a bodyweight loss over 10%, and late (i.e., after more than 30 days) deterioration of mice health was witnessed in all three groups (i.e., vehicle, T-Dxd, and T-DM1; Figure 5C and Supplementary Information). Finally, no significant differences in blood cell counts were measured in any of the four groups tested (see Supplementary Information). Overall, these results illustrate and confirm the strong potential of vincristine immunoconjugates as effective ADC for in vivo anti-cancer applications.

## Conclusions

In this paper, we reported an improved method for the synthesis of novel antibody-vincristine conjugates, with tuneable drug-to-antibody ratios. Homogeneous trastuzumab-vincristine conjugates were shown to display a high and selective cytotoxic effect in HER2⁺ cell lines, with $IC_{50}$ values a 100-fold lower than that of the free alkaloid. This potency of

trastuzumab-vincristine conjugates is comparable to the standard ADC of the field, incorporating highly cytotoxic payloads such as DM1 and MMAE, and suggests that they may present a better therapeutic index than other ADCs containing microtubule polymerization inhibitors. This was reinforced by in vivo assays in xenografted mice, which revealed a remarkable antitumor activity and no tumour relapse nor metastasis or apparent toxicity at therapeutic doses. In the attempt of finding new cytotoxic payloads for ADC applications, this work proves that conventional chemotherapeutic drugs may represent an easily accessible and abundant source of innovation.

## Experimental Section

**General methods.** All reagents were obtained from commercial sources and used without prior purification. Dry solvents were obtained from Merck. All reactions were carried out under an atmosphere of argon in flame-dried glassware with magnetic stirring. Air and/or moisture-sensitive liquids were transferred via a syringe. Concentration in vacuo refers to distillation on a Büchi rotary evaporator, and where appropriate, under high vacuum. Volume ratios (v/v) are indicated when referring to mixtures of solvents (e.g. DCM/MeOH 95:5). **Analytical thin layer chromatography (TLC)** was performed using plates cut from aluminum sheets (silica gel 60 F254, Merck, Ref. 1.05554). Visualization was achieved under a 254 or 365 nm UV light and by immersion in an appropriate staining solution. **Column chromatography** was carried out with silica gel (Geduran® Si 60, 40-63 µm) purchased from Merck. All compounds are >95% pure as determined by HPLC analysis (see below 'Analytical HPLC-MS' for small molecules and 'SEC-nMS' for antibody conjugates).

**Biomolecules.** All enzymes, reagents and solvents were obtained from commercial sources – Sigma Aldrich France, Fischer Scientific France, VWR France, New England Biolabs –, and used without prior purification. Monoclonal antibodies were provided by the Institut de Cancérologie Strasbourg Europe (Strasbourg, France). Commercial ADC T-DM1 and T-Dxd (99.74% and 99.40% pure by SEC-HPLC, respectively), and mc-Val-Cit-PAB-MMAE were purchased from MedChemExpress. Sterile human and mouse plasma in sodium citrate anticoagulant from a pool of healthy donors and BALB/c mice, respectively, were purchased from Neo Biotech (Nanterre, France). Concentrations in antibody solutions were determined by UV absorbance using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Illkirch, France) at 280 nm at ambient temperature. Sample buffer (DPBS, calcium and magnesium free, Merck, Ref. D8537-6X500ML) was used as blank for baseline correction. In case of impaired absorbance at 280 nm or unknown extinction coefficient, BCA assay was performed using a BCA Protein Assay Kit (Thermo Fisher Scientific, Illkirch, France, Ref. 23225), using BSA diluted in PBS (1X, pH 7.5) as ladder concentration. Incubation during reduction and conjugation experiments took place either in an Eppendorf thermomixer comfort (catalog #5355) or in a digital heated shaker dry bath (Ref.: 88880027).
**Biomolecules purification.** Antibody conjugates were purified by gel filtration chromatography either on Bio-spin P-6 columns obtained from Bio-rad (Hercules, U.S.A) or on Zeba™ Spin Desalting Columns, 7K MWCO, 0.5 mL (Thermo Fisher Scientific, Pierce Biotechnology, USA). Vivaspin micro-concentrators (500 µL, 50 kDa, 30 kDa, 10 kDa and 3 kDa cutoff) from Sartorius (Gottingen, Germany) were used for buffer exchange. Antibody deglycosylation was achieved by incubating Remove-iT® Endo S (New England Biolabs, Ipswich, USA) with protein samples at 10 nL/µg.

**Cell lines & cell culture.** All the cell lines used in this study were purchased from ATCC and are listed in Table S1 with their characteristics. SKBR3, BT474 and NCI-N87 cells were cultured in 4.5 g/L glucose DMEM (Gibco Life Technologies 41965-062) supplemented with 10% heat inactivated fetal calf serum (FBS) and 1% gentamicin. MDA-MB-231 cells were cultured in RPMI 1640 (Gibco 21875-034) supplemented with 10% fetal calf serum (FBS) and 1% gentamicin. MCF7 cells were cultured in 1 g/L glucose DMEM (Gibco Life Technologies 31885-049) supplemented with 10% fetal calf serum (FBS) and 1% gentamicin. Cells were maintained at 37 °C in a humidified 5% CO₂ and 20% O₂ atmosphere.

**Cell viability assays. Measure of ADCs' activity.** Cells were seeded in 96-well plates at a density of 2 000 cells/well for MDA-MB-231 cells and 5 000 cells/well for all the other cell lines and allowed to grow under normal oxygen conditions (20% O₂). Cells were treated with the appropriate compound 24 h after plating and maintained at 37 °C in a humidified 5% CO₂ and 20% O₂ atmosphere. Cell viability was measured 72 h or 120 h later with the WST-1 cell proliferation assay (Takara®) according to the manufacturer's instructions. To represent the relative cell proliferation rate, results were normalized against the untreated condition.

**Measure of bystander effect.** LNCaP FGC cells were seeded in 96-well plates at a density of 25 000 cells/well, SKBR3 EGFP cells at 7 000 cells/well, and in co-culture conditions, LNCaP FGC and SKBR3 EGFP cells were plated at densities of 1 500 cells/well and 5 000 cells/well, respectively. Cells were cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were allowed to adhere for 24 h and were then treated with the indicated concentrations of the J591-based ADCs. Each condition and ADC concentration was tested in triplicate wells. Cell viability was measured 96 h post-treatment using the alamarBlue reagent, following the manufacturer's instructions. Fluorescence was recorded using a PerkinElmer Victor X5 plate reader with an excitation filter of 531/25 nm and an emission filter of 595/60 nm. Results were normalized against the untreated control condition.

**Flow cytometry.** Cells from parallel plates used in the J591 ADC viability studies, including both individual cell lines and co-culture conditions, were processed for flow cytometry analysis. Cells were first washed with PBS, and any remaining attached cells were detached using 5 mM EDTA. After detachment, cells were washed again in PBS. The total quantity of cells, as well as EGFP expression, were analyzed using an Attune NxT flow

cytometer, with EGFP detection performed using a blue laser and a 530/30 nm emission filter.

**MS quantification of free drug in plasma.** Calibration curves were established by diluting known amounts (from 10 pmol to 1 nmol) of vincristine sulfate in water (1 µL) in human or mouse plasma (49 µL). Proteins from the biological fluid were precipitated by addition of 5 times the volume (250 µL) of cold acetone. Vincristine was recovered in the soluble fraction after centrifugation for 5 min at 12 000 x g (extraction yield 68% in mouse plasma and 40% in human plasma). Acetone was evaporated under argon flux and the residue was dissolved in 50 µL of a 1:1 (v/v) mixture of acetonitrile/water before being analyzed by LC-MS. To enhance detection sensitivity, a selected ion recording (SIR) method was set at m/z = 413 in positive ionization mode corresponding to [M + 2H]²⁺, the most abundant adduct detected. Under these analytical conditions, a lower limit of quantification (LLOQ) of 22 pmol was determined in mouse plasma, and a LLOQ of 37 pmol in human plasma, respectively. Then, T-VCR 6b (DAR = 8.5) was diluted to a final concentration of 370 µg/mL and 630 µg/mL in mouse and human plasma respectively and incubated at 37 °C for 96 h. Plasma samples (50 µL) were collected at different time points, and treated as detailed above before being analyzed by LC-MS with the dedicated SIR method.

**In vivo assays.** The right flanks of a cohort of 32 seven-week-old immunodeficient NOD-SCID mice were injected subcutaneously with SKBR3 breast cancer cells and 25% Matrigel® HC (Corning, ref. 354248) (5 x 10⁶ cells/200 µL, 81% viability as determined with "count and viability" kit, Muse®, Millipore). Treatment was initiated when the tumors reached an average size of 140 mm³, ca. 8 weeks following injection of SKBR3 cells. Four homogeneous groups of 5 to 6 animals were then formed. Each group received a single intravenous injection (5 mg.kg⁻¹) of the tested compounds: trastuzumab-vincristine T-VCR 6b, trastuzumab-embansine T-DM1, trastuzumab-deruxtecan T-Dxd, or vehicle (PBS 1X). Tumor growth was monitored using a caliper (Helios Preisser, ref. 821498) every 2-3 days. Measurements of tumor volume are presented as mean +/- SEM. Statistical analysis for tumor removal proportions (% of mice without complete tumor regression) and survival curves (% of surviving mice as a function of time postinjection) was performed using a Log-rank (Mantel-Cox) test with GraphPad Prism 7.0. Data are considered as statistically reliable when p < 0.05 (*) and p < 0.01 (**). Blood cell counts including white blood cells (WBC), lymphocytes and hematocrit (HCT) were measured on day 0, 1, 3, 7 and 21 following intravenous injection of ADC compounds (Kaslstein, Fully Auto Vet Hematology Analyzer, Model YR05122-Plus). Animal welfare was monitored each day to highlight any behavior trouble, pain or bodyweight loss according to rodent protection tests based on the APAFIS procedure #47506-2024021309116652 validated by the Strasbourg Animal Experimentation Ethics Committee (Comité d'éthique en matière d'expérimentation animale de Strasbourg, CREMEAS) and the French Ministry of Higher Education and Research (Ministère de l'enseignement supérieur et de la recherche, MESR).
**Other statistical analyses.** All *in vitro* experiments were performed at least in 3 independent biological replicates, unless otherwise indicated. Curve fits were obtained and means, SEM and IC₅₀ were calculated and analyzed using the GraphPad Prism software.

**Instrumentation and associated methods.**

¹H and ¹³C NMR spectra were recorded at 23 °C on Bruker Advance III - 400 MHz / 500 MHz spectrometers. Chemical shifts are reported in parts per million (δ) and calibrated using residual nondeuterated solvent. Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, app = apparent), coupling constant (J, Hz), integration and assignment for ¹H NMR data.

Analytical HPLC-MS analyses were carried out on Waters ARC separations module equipped with Waters 2998 PDA UV detector, Waters Acquity QDa mass detector and XBridge®, 3.5 µm, C18, 50 x 4.6 mm column. The flow rate was of 1 mL/min and the solvent system was composed as follows: solvent A: 0.05% TFA in water; solvent B: acetonitrile. The gradient run was: 0 to 5 min. – 5% to 95% B; 5 to 6 min. – 95% B; 6 to 7 min. – 5% B. Mass detector was operated in positive MS Scan mode with 600 °C probe temperature, 1.5 kV capillary voltage and 10 V cone voltage.

High resolution mass spectra (HRMS) were obtained using an Agilent Q-TOF 6520.

**Mass Spectrometry Analyses**

* **Native Mass Spectrometry (native-MS) by direct infusion mode:** After buffer exchange as described previously by Vivaspin micro-concentrators with NH₄OAc at 150 mM and pH 6.9, all samples were injected at 10 µM thanks to an automated chip based nanoESI device (TriVersa NanoMate, Advion; USA) coupled to a time of flight (TOF) mass spectrometer (LCT, Micromass/Waters, UK). The instrument was operated in positive mode, with a sample cone voltage of 120 V and an interface pressure set at 6 mbar to ensure an optimized desolvation and transmission without breaking the non-covalent interactions of the proteins. Acquisitions of mass spectra were carried out over an m/z range of 1 000 – 10 000 with a 4 s scan time. External calibration was performed using singly charged ions produced by a 2 g/L solution of cesium iodide in isopropanol/water (50:50 v/v).

* **Size Exclusion Chromatography coupled to native Mass Spectrometry (SEC-nMS).** For each sample, a volume corresponding to a range of 10 to 50 µg was injected on a BioAccord LC-MS system (Waters, Manchester, UK) for online SEC-native MS analysis. The system configuration includes an ACQUITY UPLC I-Class PLUS with an ACQUITY RDa detector (composed of an electrospray ionisation source associated to a time-of-flight mass analyser) controlled by the compliance-ready UNIFI Scientific Information System software platform. The samples were loaded onto the ACQUITY UPLC Protein BEH SEC column (2.1 x 150 mm, 1.7 µm particle size, 200 Å pore size) set at room

temperature using an isocratic elution of 50 mM ammonium acetate (NH₄OAc) at pH 6.9. A flow rate of 100 µL/min for 6.0 minutes of isocratic mode was used. The ACQUITY RDa Detector was operated in full scan mode with a positive polarity at a scan rate of 2 Hz for the high mass range (400 – 7,000 m/z). Concerning the ESI source parameters, the capillary voltage, the cone voltage and the gas desolvation temperature were fixed at 1.50 kV, 160 V and 250 °C, respectively. Additionally, the intelligent data capture was turned off and an event was activated at 4 min. (divert valve towards wastes) to avoid salts into the mass spectrometer. The ACQUITY RDa Waters connect LockMass Kit (186009298) was used as the LockSpray compound (Leucine enkephalin, caffeine and 1-pentasesulfonic acid) for real-time mass correction. The ACQUITY RDa Detector Calibrant and Wash Kit (186009013) was used to automatically calibrate the mass spectrometer before acquisition. The solution is a mixture of sodium iodide and rubidium iodide in 80:20 isopropanol/water solvent.

* **Data processing.** MS data interpretations were performed using MassLynx V4.1 (Waters, Manchester, UK) for the LCT analyses and using UNIFI v3.1.0.16 (Waters, Manchester, UK) for the BioAccord analyses. The average drug-to-antibody ratio (av. DAR) values were calculated based on the relative peak intensities measured from the deconvolution of each mass spectrum using the equation below:

$$av.\,DAR = \frac{\sum_{k=0}^{n} I_k}{\sum_{k=0}^{n} I_k}$$

Where k is the number of modifications and I_k is the relative peak intensity of species.

**Ancillary information**

**Author contributions**

A. B.: Data curation; Formal analysis; Investigation; Methodology; Project administration; Validation; Visualization; Writing – original draft

J. M.: Data curation; Formal analysis; Investigation; Methodology; Project administration; Validation; Visualization; Writing – original draft

H. C.: Data curation; Formal analysis; Methodology; Validation; Visualization; Writing – original draft

L. M. d. S.: Data curation; Formal analysis; Visualization; Writing – original draft

F. Dau.: Data curation; Formal analysis; Investigation; Methodology; Resources; Validation; Visualization

S. E.: Data curation; Formal analysis; Investigation; Methodology; Validation; Visualization;

S. C.: Funding acquisition; Methodology; Project administration; Resources; Supervision; Writing – review & editing

O. H.-A.: Methodology; Project administration; Resources; Supervision

C. S.: Data curation; Formal analysis; Investigation; Validation

I. D.: Visualization; Writing – original draft

S. K.: Resources; Writing – review & editing
A. D.: Resources; Writing – review & editing

F. Dan.: Funding acquisition; Methodology; Project administration; Resources; Supervision

A.W.: Funding acquisition; Methodology; Project administration; Resources; Supervision

M. Z.: Conceptualisation; Data curation; Formal analysis; Funding acquisition; Methodology; Project administration; Resources; Supervision; Validation; Visualization; Resources; Writing – review & editing

G. C.: Conceptualisation; Data curation; Formal analysis; Funding acquisition; Methodology; Project administration; Resources; Supervision; Validation; Visualization; Resources; Writing – review & editing

## Conflicts of interest

There are no conflicts to declare.

## Supporting Information Availability

Experimental data supporting this article have been included as a PDF file containing: solubility data, experimental procedures, procedures for the synthesis of compounds **2, 3, 8a, 8b**, copies of ¹H and ¹³C NMR spectra, HPLC chromatogram and full characterization of **3**, details of in vitro and in vivo assays. Molecular Formula Strings has been included as a CSV file.

## Acknowledgements

This work was supported by the LabEx Medalis and the Interdisciplinary Thematic Institute IMS (Institut du Médicament Strasbourg), as part of the ITI 2021-2028 supported by IdEx Unistra (ANR-10-IDEX-0002) and the SFRI-STRAT'US project (ANR-20-SFRI-0012), the "Agence Nationale de la Recherche" (Conformabs – ANR-21-CE29-0009-01), the CNRS, the University of Strasbourg, and the French Proteomic Infrastructure (ProFI; ANR-10-INBS-08-03). A.D. further acknowledges support from the Labelisation Ligue contre le cancer.

## Abbreviations used

ADC, antibody-drug conjugates; Cit, citrulline; DAR, drug-to-antibody ratios; DIPEA, N,N-diisopropylethylamine; DM1, mertansine; Dxd, deruxtecan; HER2, Human Epidermal Growth Factor Receptor-2; mAb, monoclonal antibody; mc, maleimidocaproyl; MMAE, monomethyl auristatin E; NOD-SCID, Non-obese diabetic-severe combined immunodeficiency; PAB, p-aminobenzyl; TBAI, tetrabutylammonium iodide; TCEP, tris(2-carboxyethyl)phosphine; TROP2, trophoblast cell-surface antigen 2; VCR, vincristine

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