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. 2019 Dec 3;10(12):1674–1679. doi: 10.1021/acsmedchemlett.9b00468

Discovery of Potent and Selective Antibody–Drug Conjugates with Eg5 Inhibitors through Linker and Payload Optimization

Alexei S Karpov †, Cristina M Nieto-Oberhuber †, Tinya Abrams §, Edwige Beng-Louka †, Enrique Blanco †, Sylvie Chamoin †, Patrick Chene †, Isabelle Dacquignies †, Dylan Daniel ‡, Michael P Dillon §, Lionel Doumampouom-Metoul †, Nikolaos Drosos, Pavel Fedoseev †, Markus Furegati †, Brian Granda §, Robert M Grotzfeld †, Suzanna Hess Clark §, Emilie Joly †, Darryl Jones †, Marion Lacaud-Baumlin †, Stephanie Lagasse-Guerro †, Edward G Lorenzana ‡, William Mallet ‡, Piotr Martyniuk †, Andreas L Marzinzik †, Yannick Mesrouze †, Sandro Nocito †, Yoko Oei ‡, Francesca Perruccio †, Grazia Piizzi †, Etienne Richard †, Patrick J Rudewicz ‡, Patrick Schindler †, Mélanie Velay †, Kristine Venstrom ‡, Peiyin Wang ‡, Mauro Zurini †, Marc Lafrance †,§,*
PMCID: PMC6912867  PMID: 31857845

Abstract

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Targeted antimitotic agents are a promising class of anticancer therapies. Herein, we describe the development of a potent and selective antimitotic Eg5 inhibitor based antibody–drug conjugate (ADC). Preliminary studies were performed using proprietary Eg5 inhibitors which were conjugated onto a HER2-targeting antibody using maleimido caproyl valine-citrulline para-amino benzocarbamate, or MC-VC-PABC cleavable linker. However, the resulting ADCs lacked antigen-specificity in vivo, probably from premature release of the payload. Second-generation ADCs were then developed, using noncleavable linkers, and the resulting conjugates (ADC-4 and ADC-10) led to in vivo efficacy in an HER-2 expressing (SK-OV-3ip) mouse xenograft model while ADC-11 led to in vivo efficacy in an anti-c-KIT (NCI-H526) mouse xenograft model in a target-dependent manner.

Keywords: Antibody−drug conjugate (ADC); HER2; c-KIT; antimitotic; kinesin spindle protein (Eg5, KSP1, KIF11)

In the most common form, an antibody–drug conjugate (ADC) comprises three components: an antibody that targets a cell-surface antigen expressed by cancer cells, to which is attached via a linker a cytotoxic small molecule (payload) with the aim of minimizing negative effects to healthy tissues.1,2 The antibody specifically recognizes an overly expressed antigen receptor on the cells of interest, generating the desired selectivity. Second, a cleavable or noncleavable linker is employed to attach the payload onto the antibody. Upon lysosomal catabolism, cleavable linkers release the free permeable payload,3 which could exert a subsequent bystander killing effect. Alternatively, payloads attached via noncleavable linkers are released as a charged adduct following catabolism, preventing a bystander killing effect.4 Lastly, the highly active payload is responsible for the desired biological effect. To this date, ADCs have been mostly studied with oncologic payloads, including the recent clinical approval of four ADCs (Mylotarg,5 Adcetris,6 Kadcyla,7 and Besponsa8), but recent examples targeting bacterial infections9 and immunology10,11 have also emerged. Despite the challenges of dealing with the off-target toxicities12 arising from conjugates with antimitotic (auristatins6 maytansines7 and tubulysin13−15) and DNA alkylating agents,5,8,16,17 these payload classes are still dominating the more than 150 ADCs currently undergoing clinical studies.18 The development of payloads with novel mechanism of action (MoA) is required for the successful expansion of this therapeutic approach to a broader set of tumor types. Recently, alternative MoA payloads have been reported including DNA intercalators,19,20 RNA-polymerase II inhibitors,21 RNA splicing inhibitors,22 DNA topoisomerase inhibitor,23,24 as well as NAMPT inhibitors25,26 to address this challenge.

In pursuit of a novel ADC platform that provides improved efficacy and tolerability, we sought potential payloads that act through more selective mechanisms. Eg5 (KSP-5, kinesin spindle protein-5, KIF11) is a motor protein that is required for bipolar spindle formation during mitosis.27 Several Eg5 inhibitors have been developed and entered clinical assessment, e.g. NVP-BQS481, SB715992 (Ispinesib), and ARRY-520 (Filanesib) (Figure 1).28 These molecules have shown high preclinical antitumor potency and are generally well-tolerated but suffer from fully reversible neutropenia, which is expected of antimitotic agents.29,30 We envisioned that a targeted therapy based on ADCs would significantly improve the therapeutic index.31−33

Figure 1.

Figure 1

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Examples of Eg5 inhibitors in clinical trials and internal examples of Eg5 inhibitors.

NVP-BQS481 and other analogs synthesized during the Novartis discovery program, e.g. compound 2 (Figure 1),30 were selected as a starting point for our Eg5 ADC efforts. We began our efforts using cleavable linkers such as the commonly employed Cathepsin-cleavable linker MC-ValCit-PABC (L1). With this approach, intracellular catabolism generates the parent payload, and cellular activity is directly related to the activity of that payload, which simplifies the overall ADC design. The use of linkers that are cleaved by lysosomal proteases such as cathepsin B also enhances the efficiency of payload release. We synthesized the initial linker-payload (LP-1) by attaching the linker MC-ValCit-PABC (L1) onto the free basic amine of the payload 1. Conjugation was performed using maleimide chemistry to the interchain disulfide bonds of a HER2-targeting antibody to give ADC-1 (Table 1).34

Table 1. Comparison of in Vitro Cytotoxicity of Eg5 Inhibitor Payloads 1-2 and Their HER2-ADCs Using a Cleavable Linker.

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ADC Linker-Payload Target antigen DAR Aggr. (%) SK-OV-3ip (HER2+, nM) MDA-MB-231-M16 (HER2+, nM) MDA-MB-468 (HER2–, nM)
ADC-1 LP-1 HER2 2.8 40.0 n.d. 27 >3000
ADC-2 LP-2 HER2 4.0 4.0 6.4 17 >3000
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Although ADC-1 suffered from high aggregation (40%), it demonstrated good selectivity between HER2+ cell lines vs HER2– cell lines (Table 1). Modification of the payload 1 with the aim to reduce lipophilicity while retaining high potency led to payload 2 (cLogP 4.7 vs 3.1, respectively). In particular, the primary amine function in 1 was replaced with a pyrrolidine moiety, and the tert-butyl group was replaced with a more hydrophilic tetrahydropyran motif. Following the same transformation and conjugation onto the HER2-antibody led to an equipotent but much lower aggregating (4%) conjugate, ADC-2 (Table 1).

To assess the specificity of our ADC, a negative control was generated. Conjugation of LP-2 with a nontargeted IgG isotype-matched control antibody gave ADC-3.35ADC-2 as well as its isotype-matched control ADC-3 were then evaluated in vivo in an ovarian SK-OV-3ip xenograft tumor model (high HER2) in mice with a single 3 mg/kg dose (Figure 2). Administration of ADC-2 induced long-term efficacy for approximately 30 days. Unfortunately, a significant amount of antigen-independent activity was observed for the nonbinding control (ADC-3). A plausible explanation is premature release of the payload due to linker cleavage by mouse carboxylesterase.36

Figure 2.

Figure 2

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In vivo antitumor effect of Eg5 ADC-2 and ADC-3 with cleavable linkers in the HER2+ SK-OV-3ip model in mice.

Premature release of the payload can be attenuated by switching to more stable noncleavable linkers. For this approach, we must identify the optimal attachment point on the Eg5 inhibitor from which the stable linker can be attached without interfering with its binding and function. Figure 3 shows the docking structure for compound 2, where the aromatic rings are deeply buried into the binding site. In contrast, the lactate group (Scaffold A), the tetrahydropyran (Scaffold B), and the pyrrolidine (Scaffold C) motifs are solvent exposed and offer appropriate exit vectors to attach the linker.

Figure 3.

Figure 3

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Docked structure of compound 2 within Eg5 protein pocket.

Three different exit vectors were identified. In Scaffold A (3), the linker is attached to a suitable spacer extending from the central secondary amine. In Scaffold B (4), the linker is attached through elongation of the t-Bu group. Finally, in Scaffold C (5), the (R)-fluoropyrrolidine is replaced by a (S)-hydroxypyrrolidine where the linker can be attached.

Following lead optimization of the 3 exit vectors with concomitant tuning of the linker properties, we identified different suitable LPs combinations which were conjugated onto 2 different antibodies targeting HER2 and c-KIT.34 Both HER2 and c-KIT are highly expressed cell surface targets, and antibodies targeting these receptors can efficiently internalize and serve as efficient delivery vehicles for ADC payloads. The summary of the results of the corresponding ADCs (ADC-4 to ADC-13) is found in Table 2. In general, all the conjugates retained a low level of aggregation (0.5–3.6%), with a drug-to-antibody ratio ranging between 3.4 and 5.8. All the conjugates were first tested in vitro on either HER2+ (SK-OV-3ip) or HER2– cells (MDA-MB-468) and on c-KIT+ cells (NCI-H526). For the HER2 conjugates, EC50 values ranged from 7.6 to 43.7 nM and have similar potency to the conjugates with a cleavable linker. The c-KIT conjugates were generally slightly less potent (12.8–4528 nM) with ADC-13 being inactive. Interestingly, the potency of the conjugates ranked differently depending on the antibody used. For HER2, all the conjugates had comparable potency whereas for c-KIT conjugates, ADC-11 was found to be the significantly most potent conjugate.

Table 2. Summary of in Vitro Cytotoxicity of Representative Payloads and ADCs with Stable Linkers.

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ADC linker- payload Target antigen DAR Aggr. (%) SK-OV-3ip (Her2+, nM) MDA-MB-468 (Her2–, nM) NCI-H526 (c-KIT+, nM)
ADC-4 LP-3 HER2 5.8 0.7 14.1 3423 n.d.
ADC-5 LP-3 c-KIT 3.4 1.6 n.d. >9000 286
ADC-6 LP-4 HER2 5.2 1.4 25.2 8171 n.d.
ADC-7 LP-4 c-KIT 3.6 0.9 n.d. >9000 29.8
ADC-8 LP-5 HER2 4.0 2.1 7.6 3429 n.d.
ADC-9 LP-5 c-KIT 4.2 0.7 n.d. 7382 42.4
ADC-10 LP-6 HER2 4.4 2.6 8.2 5620 n.d.
ADC-11 LP-6 c-KIT 4.1 0.5 n.d. 6612 12.8
ADC-12 LP-7 HER2 3.9 3.6 43.7 5672 n.d.
ADC-13 LP-7 c-KIT 3.7 1.0 n.d. 8273 4528
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Several ADCs demonstrated good in vitro potency and were therefore selected for in vivo studies. Again, nontargeting IgG isotype-matched negative control ADC was generated by conjugation to LP-3 with the nonbinding IgG antibody to give ADC-14.37 An initial in vivo dose assessment was performed using ADC-4 (in SK-OV-3ip model; n = 9 mice/group) and ADC-5 (in NCI-H526 model; n = 5 mice/group) at 5 and 10 mg/kg (Figure 4) and compared to the negative control ADC-14 at 10 mg/kg. A single 10 mg/kg intravenous administration of ADC-4 and ADC-5 induced tumor stasis for approximately 2 weeks before tumors exhibited regrowth, while the efficacy was shorter-lived at the 5 mg/kg dose level. Further, no significant amount of antigen-independent activity was observed for the nonbinding control (ADC-14). In vivo mouse model efficacy of the ADCs was then assessed at 5 mg/kg (Figure 5) to achieve differentiation of the different ADCs. ADC-4, ADC-8, and ADC-10 were assessed and compared with ado-trastuzumab emtansine (Kadcyla) (5 mg/kg) in the SK-OV-3ip model.37 A single intravenous administration of ADC-4 and ADC-10 led to tumor stasis for 3 weeks as well as displayed superiority over ado-trastuzumab emtansine. As for c-KIT+ model NCI-H526, ADC-5, ADC-7, ADC-9, ADC-11, and ADC-13 were assessed with only ADC-11 being able to induce tumor stasis at submaximal efficacious dose for 2 weeks.

Figure 4.

Figure 4

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In vivo dose assessment of the antitumor efficacy of Eg5 ADC-4 in the HER2+ (SK-OV-3ip model) and ADC-5 in the c-KIT+ (NCI-H526 model) against ADC-14 in mice.

Figure 5.

Figure 5

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Comparison of in vivo antitumor effect of ADCs in HER2+ (SK-OV-3ip model) and c-KIT+ (NCI-H526 model) in mice.

Further assessment of ADC-4, ADC-10, and ADC-11 revealed a PK profile showing an expected low volume of distribution, low clearance, and a long half-life. Their integrity was maintained with the exception of the hydrolysis of the succinimide over the course of days. The most efficacious ADC-4 and ADC-10 were also the most stable in vivo in rats in comparison to ADC-8, which was less efficacious and also less stable (Figure 6). A reduction of DAR was observed (up to 70%) after 20 days.

Figure 6.

Figure 6

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In-vivo DAR evolution (in %) of ADC-4, ADC-8, and ADC-10. Each data point is an average of 3 biological replicates (3 rats) as well as a technical duplicate for each run (rats).

Finally, an in vivo crossover experiment was performed to demonstrate the specificity of the conjugates (Figure 7).37 Mice were implanted in contralateral flanks with c-KIT expressing NCI-H526 cells as well as HER2 expressing SK-OV-3ip cells.

Figure 7.

Figure 7

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Crossover in vivo experiment of ADC-4 and ADC-5 for HER2+ SK-OV-3ip and c-KIT+ model, respectively, in mice.

Mice were then treated with ADC-4, ADC-5, and nonbinding ADC-14 as a negative control at DAR matched dose levels. Antitumor efficacy was observed only for the tumor expressing the antigen recognized by the dosed ADC. This result excludes the hypothesis that ADC efficacy is due to target-independent generation of an active catabolite. The most likely mechanism is that the catabolite only acts on the cell of origin, although some contribution to local “bystander” activity cannot be excluded.

In summary, a potent Eg5 inhibitor developed from a small-molecule program was optimized as a suitable and highly potent ADC payload. Antigen-independent activity with cleavable linker ADCs prompted us to investigate different strategies of stable linkers. A variety of suitable attachment points on the payload were compared leading to the optimized ADC-4, ADC-10 (against HER2+ cell lines), and ADC-11 (against c-KIT+ cell lines). These ADCs have demonstrated an expected PK profile and excellent specificity. We have shown evidence that Eg5 inhibitor ADCs have the potential for superior in vivo efficacy compared to ado-trastuzumab emtansine (Kadcyla). Further profiling and evaluations of these ADCs are currently underway.

Acknowledgments

We are grateful to Paul Barsanti for scientific discussion and Regis Denay, Christophe Bury, Thierry Besson, and Thomas Wolf for their excellent technical assistance.

Glossary

Abbreviations

ADC

Antibody–drug conjugate

Eg5

kinesin-like protein 1

Her

herceptin

HER2

human epidermal growth factor receptor 2

DNA

deoxyribonucleic acid

MoA

mechanism of action

RNA

ribonucleic acid

NAMPT

nicotinamide phosphoribosyltransferase

MC-ValCit-PABC

maleimido caproyl valine-citrulline para-amino benzylcarbamate

DAR

drug-to-antibody ratio

Mal

maleimide

n.d.

not determined

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00468.

  • Complete experimental procedures and analytical characterization of novel compounds, bioconjugation methods, in vitro cytotoxicity, and in vivo efficacy data (PDF)

Author Present Address

∥ (D.D.) CytomX Therapeutics, 151 Oyster Point Blvd, Suite 400, South San Francisco, CA 94080, United States.

Author Present Address

⊥ (M.P.D.) Ideaya Biosciences, 7000 Shoreline Court, Suite 350, South San Francisco, CA 94080, United States.

Author Present Address

# (E.G.L.) Revolution Medicines, 700 Saginaw Drive Redwood City, CA 94063, United States.

Author Present Address

∇ (W.M.) Bolt Biotherapeutics, 640 Galveston Drive, Redwood City, CA 94063, United States.

Author Present Address

□ (Y.O. and P.W.) Genetech Inc., 1 DNA Way, South San Francisco, CA 94080, United States.

Author Present Address

○ (G.P.) Cygnal Therapeutics, 325 Vassar Street, Cambridge, MA 02138, United States.

Author Contributions

The manuscript was written through contributions of all authors.

The authors declare no competing financial interest.

Supplementary Material

ml9b00468_si_001.pdf (1.1MB, pdf)

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Supplementary Materials

ml9b00468_si_001.pdf (1.1MB, pdf)

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