Recent Advances in the Development of New Auristatins: Structural

Review pubs.acs.org/molecularpharmaceutics

Recent Advances in the Development of New Auristatins: Structural Modifications and Application in Antibody Drug Conjugates Andreas Maderna* and Carolyn A. Leverett Pfizer Worldwide Research and Development, Worldwide Medicinal Chemistry, Oncology, Eastern Point Road, Groton, Connecticut 06340, United States

ABSTRACT: Dolastatin 10 is a powerful antineoplastic agent and microtubule inhibitor that was discovered by Pettit et al. and published in 1987. Since then, many research groups have engaged in SAR studies of synthetic analogues, termed “auristatins”. It was eventually discovered that auristatins are of great value as payloads in antibody drug conjugates (ADCs), which led to the FDA-approved ADC brentuximab vedotin (Seattle Genetics). Currently, over 30 ADCs in clinical trials employ auristatins as payloads, and there is a great interest in the research community, both on academic and industrial sides, to further study these analogues. This review will provide an overview of the recent advancements in auristatin development spanning a time frame of about the past ten years. The main focus will be to describe structural changes made to the auristatin peptide and their resulting biological activities in tumor cell proliferation assays. Selected ADC examples will also be described. KEYWORDS: dolastatin 10, auristatins, structural modifications, antibody drug conjugates site.8,9 In addition to the effect of dolastatin 10 (1) on tubulin dynamics, leading to mitotic cell cycle arrest and apoptosis, it was soon recognized that dolastatin 10 (1) and related synthetic auristatin analogues also have strong antivascular effects.10,11 The immense potency and potential of dolastatin 10 (1) for use as a cancer chemotherapeutic agent has spurred structure−activity relationship (SAR) studies of these highly potent cytotoxins, as well as investigations of their activity profiles.5,12,13 Figure 2 shows key analogues reported by Pettit et al. during the early development of this compound class.5,12,14,15 SAR studies of Auristatin-PE (2) has also been reported by Miyazaki et al.13 Initially, dolastatin 10 (1) itself (Figure 1) was advanced into clinical trials, but despite encouraging preclinical efficacy data, no appreciable therapeutic index could be attained due to its significant toxicity at the maximum tolerated dose (MTD).16−22 Several analogues with C-terminal modifications were identified

1. INTRODUCTION The discovery of dolastatin 10 (1) by Pettit et al. is a landmark achievement in the development of highly cytotoxic antineoplastic agents for cancer chemotherapy.1 In his pioneering work, Pettit et al. isolated dolastatin 10 (1) from the sea hare Dolabella auricularia, determined its precise chemical structure, and enabled its first total synthesis.2 A remarkable feature of this shell-less mollusk is its ability to sequester and store secondary metabolites from dietary sources in their digestive system that serve as a chemical defense mechanism against predators.3,4 In fact, a whole suite of cytotoxic peptides was isolated from these animals, of which dolastatin 10 (1) is the most potent analogue.5 It was soon recognized that the extraordinary cytotoxicity of dolastatin 10 (1), exhibiting picomolar GI50 values in most cancer cell proliferation assays, is caused by its ability to strongly inhibit microtubule assembly and tubulin-dependent GTP hydrolysis, resulting in cell cycle arrest and apoptosis.7 Recent work has revealed the precise binding mode of dolastatin 10 (1) and its synthetic analogues (termed “auristatins”) at the α,β-tubulin interphase, with the N-terminal end being in proximity to the vinca binding domain and the Cterminal end residing near the exchangeable GTP binding © XXXX American Chemical Society

Special Issue: Antibody-Drug Conjugates Received: November 16, 2014 Revised: February 7, 2015 Accepted: February 16, 2015

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DOI: 10.1021/mp500762u Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Figure 1. Structure of dolastatin 10 (1) and its key amino acid building blocks. The GI50 value is an average of data obtained with various colon cancer cell lines.6 The N- and C-termini are represented by dolavaline and dolaphenine, respectively.

ADC is directed against CD30+ cells and uses vc-MMAE (Figure 3) as the linker-payload, with an average drug to antibody ratio (DAR) of four. The linker shown in Figure 3 is cleaved by proteases, such as cathepsin B, to release the unmodified drug. Linkers that have an engineered cleavage sequence are termed “cleavable”. It needs to be pointed out that next to the auristatins a second and equally important payload class exists, namely, the maytansinoids. These payloads, also very potent tubulin polymerization inhibitors, were successfully developed as ADC payloads with outstanding achievements by Lambert and Chari et al. at ImmunoGen. ADCs with maytansinoids include the FDA approved ADC Ado-trastuzumab emtansine (T-DM1, partnered with Genentech/Roche) for Her-2 expressing breast cancer.31 In addition, many other promising maytansine-containing ADCs are currently being developed by ImmunoGen and its partners.29 The remarkable success story of the highly efficacious brentuximab vedotin (SGN-35) ignited a second wave of research aimed toward discovery of new auristatin analogues and linker derivatives tailored for use in ADC applications. Further, it is now recognized that the true clinical value of the auristatins is based on their employment as ADC payloads rather than as systemically dosed stand-alone drugs. In addition, other targeted modalities for the selective delivery of auristatins to tumors are being considered, such as MMP-targeting prodrug analogues and albumin conjugates having targeting peptide ligands.32−36 To date, all auristatin-based ADCs currently in clinical trials use either MMAE (7), MMAF (8) (Seattle Genetics), or the recently described dolastatin 10 analogue 9 (PF-06380101, Pfizer) in which the N-terminal dolavaline is replaced by 2methylalanine (Figure 4), see section 2.3.8,37 Analogue 9 also differs from 7 and 8 in that it contains a primary amino group on the N-terminus and that it features the naturally occurring dolaphenine C-terminus. MMAF (8), which possesses a free carboxylic acid, exhibits reduced cell potency as a free payload, presumably due to its compromised cell-membrane permeability.38 Extensive research by Doronina and Senter et al. led to the successful employment of MMAF (8) as payloads in several ADC programs currently in clinical trials. In all these clinical examples MMAF (8) is attached to the antibody via a “noncleavable” linker, termed this way because lysosomal catabolism is needed to release the

Figure 2. Key synthetic dolastatin 10 (1) analogues (auristatins) first reported by Pettit et al. Mean GI50 values were obtained from quadruplicate screenings of compounds indicated against the National Cancer Institute’s full 60-cancer cell line panel.5,12

during SAR campaigns, among them auristatin-PE (2) and auristatin-PYE (3), both resulting in reduced cytotoxicity compared to its natural product analogue dolastatin 10 (1). However, despite their attenuated potency, significant adverse events were recorded in clinical trials at dose levels that were not sufficient to attain clinical efficacy.23−26 These setbacks limited the promise of dolastatin 10 (1) and its synthetic auristatin analogues for future clinical applications. An important discovery that eventually changed the outlook for auristatins was made by Miyazaki et al., who found that the removal of one N-methyl group of dolavaline at the N-terminus of dolastatin 10 (1) to give analogue 6 only slightly attenuated potency (Figure 3).13 This finding was recognized by Senter et al., who made the remarkable discovery that these auristatins having a secondary amine at their N-terminus could be attached to a linker and subsequently conjugated to monoclonal antibodies. This clever idea led to generation of highly potent and efficacious antibody drug conjugates (ADCs).27−29 Later, this pioneering work by Senter et al. at Seattle Genetics led to the discovery of the FDA approved ADC brentuximab vedotin (SGN-35), a highly efficacious ADC currently approved for the treatment of relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma.30 This B


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Figure 3. (A) N-des-Methyl analogues MMAD (monomethyl auristatin-D, 6) and MMAE (monomethyl auristatin-E, 7) retain most of the cytotoxic potential of their dolavaline counterparts. GI50 values are obtained with cell lines shown in the brackets. (B) MMAE (7) connected to the vc-linker and conjugated to an antibody via maleimide-Cys addition. The drug to antibody ratios (DAR) can vary (shown here is a DAR of 1). Proteolytic linker cleavage releases unmodified MMAE (7).27

acid residue of the conjugated antibody (in this case, cysteine), resulting in an overall released Cys-mc-MMAF species (10). In this case, binding affinity to tubulin inside cells is not compromised but does result in reduced cell-based potency when tested as a free payload due to compromised cellmembrane permeability.38 It should be noted that the term noncleavable does not indicate complete in vivo stability and that this terminology is somewhat misleading. In fact, maleimide-deconjugation reactions via a retro-Michael reaction with subsequent trapping of the regenerated maleimide by free cysteine, glutathione or albumin, is a possibility, and the transfer to Cys-34 of albumin has been particularly well documented. 39,40 It is conceivable that the same mcdeconjugation process could occur in vivo in various tissues in the presence of high concentrations of sulfur-containing nucleophiles. To what extent this might also occur for the vclinker or other maleimide-containing linker systems is unclear. Excellent work has also been performed by Senter et al. to study ADC biodistribution utilizing a dually labeled mc-MMAF containing ADC.41 This work has provided valuable insights into tissue uptake profiles of both ADC and released Cys-mcMMAF. In addition to the development of MMAF, these authors have developed several MMAF-related payloads that contain further modified ester or amide bonds on the Phecarboxylic acid.42,43 Several highly in vivo efficacious ADCs could be prepared with these modified MMAF analogues,

Figure 4. Auristatin analogues currently in clinical trials as ADC payloads.

payload species, Cys-mc-MMAF (10), inside the cell (Figure 5).38 As shown in Figure 5, after ADC catabolism the released payload species contains both the linker and the first amino C


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Figure 5. MMAF conjugated to an antibody via the maleimidocaproyl linker (“mc”-linker). This linker does not possess a proteolytic cleavage sequence and requires lysosomal degradation to release Cys-mc-MMAF (10).

Figure 6. Auristatin structural modifications described in this review.

2.1. C-Terminal Modifications. A large number of Cterminal modifications have been previously reviewed as part of early auristatin SAR campaigns.5,12,13 Since these reports, Doronina et al. described analogues of MMAF (8), in which the carboxylic acid of phenylalanine is replaced by tetrazole and phosphonate isosteres (11 and 12, Figure 7).44 Both

including examples that contain aniline functionalities suitable for C-terminal linker attachment.43 This brief review will provide an overview of the recent advancements in auristatin development spanning a time frame of about the past ten years. Major new trends in SAR will be summarized, without an exhaustive discussion of all compounds studied. The main focus will be to describe structural changes made to the auristatin peptide and their resulting biological activities in tumor cell proliferation assays. Selected ADC examples will also be described, but the main emphasis will be on payload SAR studies. Many of the studies reviewed here are taken from the patent literature and refer to preclinical ADC investigations. Current clinical trials with MMAE (7), MMAF (8), or PF-06380101 (9)-containing ADCs will not be reviewed.

2. AURISTATIN STRUCTURAL MODIFICATIONS Most studies focused initially on auristatin analogues have centered on C-terminal modifications and/or analogues with N-terminal extensions (Figure 6) without alteration of the remaining peptide core. These two approaches were likely chosen as the starting points for subsequent modifications based on earlier reported studies showing that the C-terminal amine dolaphenine could be replaced, providing analogues with good potency in cell based toxicity assays. These studies have been extensively reviewed.5,12,13 The structural modification discussed in this review will cover C-terminal modifications, N-terminal extensions, replacements of the N-terminal dolavaline, and changes to the central peptide core structure (Figure 6).

Figure 7. Tetrazole and diethylphosphonate analogues of MMAF (8).

compounds showed good activity as free payloads with H3396 breast carcinoma cells and HCT-116 colon carcinoma cells, respectively. The phosphonate ester 12 shows good cell potency, whereas the corresponding phosphonic acid derivative is significantly less active as a free payload (237−790 nM in various cell lines).44 The reduced potency can be contributed to the compromised cell permeability, a phenomenon also observed for MMAF (8), which also exhibits reduced cellbased potency as a free payload (58−380 nM in cells) due to D


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Molecular Pharmaceutics the negative charge of the carboxylate.38 However, if the negative charge is masked by an ester functionality the cell potency is restored, presumably due to increased passive cellpermeability. One example is MMAF-OMe (Figure 4, 8-OMe), which shows similar potency to MMAE (7) in cell-based cytotoxicity assays.8,38 However, in context of ADCs, the cell membrane permeability of the free payload is not a primary concern since the ADC serves as a delivery vehicle to shuttle the payload inside the cell.38 Both auristatins 11 and 12 (free acid version) provided active ADCs when conjugated to the anti-CD70 h1F6-antibody via the vc-linker, with potencies comparable to those prepared with MMAF (8) as the payload (GI50 4−10 ng/mL).44 Years after his initial studies with dolastatin 10 (1), Pettit et al. also pursued auristatins containing C-terminal aromatic phosphate and quinoline modifications (13−15, Figure 8).45,46

Figure 9. (A) Auristatin with C-terminal hydroxamate. (B) Trastuzumab ADC with C-terminal linker attachment and conjugation via lysine. Drug antibody ratio (DAR) not reported.

Figure 10. Linker attachment on the C-termini of auristatin-F related analogues leading to linker payload structure 18. The scissile bond cleaved by proteases is indicated by the arrow. Red, payload; blue, linker.

Figure 8. C-Terminal aryl phosphates and quinolines described by Pettit et al.

The described compounds contain the natural dolavaline Nterminus. The observed cell potency of 13 is remarkable despite the presence of two negative charges, potentially indicating that phosphate hydrolysis, catalyzed by phosphatases or related hydrolases, occurs in the assay medium to release the more cellmembrane-permeable auristatin phenol. However, detailed studies about phosphate cleavage in the assay medium to confirm the prodrug hypothesis was not presented.45 If confirmed, this phosphate prodrug approach would be reminiscent of that applied for combretastatin A4 phosphate.47 In addition, 13 was reported to have excellent water solubility (>236 mg/mL). Good activity was also observed for the two quinoline analogues 14 and 15, with 14 being substantially more potent. A C-terminal hydroxamic acid auristatin analogue 16 has also been reported, which has been employed in the development of the trastuzamab ADC 17 via lysine conjugation and C-terminal linker attachment (Figure 9).48 The linker used in ADC 17 contains a simple p-aminoalcohol (PABA) spacer attached to alanine and does not contain a typical amino acid dimer sequence that is known to be recognized by cathepsins.49,50 Detailed mechanistic studies were not presented regarding the nature of this linker (cleavable or noncleavable by proteases). The concept of attaching the linker on the C-terminus was first introduced by Doronina et al., who described proteolytically cleavable linkers with various amino acid sequences on the C-terminus of MMAF (8), leading to linker payload structure 18 (Figure 10).51−53 Several peptide dimers AA1-AA2 were investigated, utilizing linkage by cysteine thiol groups of the 1F6 antibody.

The prepared ADCs were directed toward CD70-antigens present on cells derived from hematologic malignancies and renal cell carcinoma. Several amino acid pairs, such as Met(D)Lys and Asn-(D)Lys, were found to provide highly in vitro potent and in vivo efficacious ADCs. In particular, some ADCs with these linker arrangements were better tolerated and more efficacious than the corresponding ADCs with N-terminal linked vc-MMAF.51 Park et al. has also explored auristatin C-terminus variations, specifically by pursuing auristatins containing an α-aminoketone C-terminus (Figure 11).54 In particular, the described Lys-conjugated ADC 19 showed good efficacy in a BT474 xenograft model at single doses from 1 to 5 mg/kg. The extent of variation allowed in the C-terminal auristatin binding domain was further exemplified by Lerchen et al., who installed sterically demanding substituents at the C-terminus, which led to potent auristatin analogues, including 20−22 (Figure 12).55 The tolerability for these significant structural changes is not surprising based on the auristatin-tubulin crystal structure analyses, which show a large solvent-exposed shallow binding cleft.8,9 However, the overall peptide confirmation is a critical component in the overall binding affinity, and any Cterminal changes must not deviate from the tubulin bound-low energy conformation. In earlier work, Senter et al. reported a set of ester analogues, all of which were attached to targeting antibodies via an acidsensitive hydrazone linker (Figure 13).27,56 E


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Figure 11. Trastuzumab ADCs with aminoketone-derived auristatins conjugated to lysine.

proliferation assays and led to significant activity in antigennegative cells. In addition, the plasma stability and in vivo tolerability of 23 was inferior to that of the vc-MMAE ADC, eventually leading to the abandonment of these hydrazonelinked ADCs and replacement with the cathepsin-cleavable vcMMAE. Regardless, the apparent efficacy with 23 on a 3 mg/kg dosage (MTD = 15 mg/kg), without observance of overt toxicity, is noteworthy and represents an early example of how C-terminal linker connections to auristatins could be successfully established.27 2.2. C-Terminal Modifications and N-Terminal Extensions. Extensive studies have been conducted by Lerchen et al., who described auristatin analogues with both N-terminal extensions and C-terminal modifications (Figure 14).57 The key design criterion that guided the SAR efforts was to maximize the intracellular residence time of the auristatins after ADC delivery. It was hypothesized that cell membrane permeability from the basolateral to the apical site [Papp (B > A)] is of critical importance for toxophores, which are released intracellularly. In this case, the lower the permeability, the longer the residence time of the payload in the cell following linker cleavage and intracellular payload release. This consequently results in more time available for interaction with the biochemical target (in this case, tubulin). The permeability was measured using the flux assay with Caco-2 cells. Compounds 24−26 (Figure 14), shown as their Cys-capped maleimide versions, are specific examples of many analogues that were prepared pursuing this design concept.57 Also notable are the hydrazide functionalities present in the linker. Structures 24−26 represent the released payload species, which include a residual cysteine attached to the N-terminus following lysosomal ADC catabolism. This is analogous to mcMMAF-type ADCs with noncleavable linkers, where Cys-mcMMAF is the released payload species, having retained tubulin binding affinity but with low passive cell-permeability (Figure 5).38 All described compounds in their study exhibit low permeability from B to A [Papp (B > A)] when tested separately as free payload species and therefore have a long residence time in the Caco-2 cells. In comparison, MMAE (7) was reported to exhibit a permeability value [Papp (B > A)] of 73 nm/s and therefore has significantly shorter residence times in the Caco-2 cells. No value for Cys-mc-MMAF was provided for comparison. It is also important to mention that this strategy prevents the potential for the “bystander effect,” which often is considered beneficial for efficacy, especially in addressing tumor heterogeneity and low antigen expression profiles across the malignant cell population.29,58 The bystander effect is enabled by the diffusion of payloads from the intracellular into the extracellular space after the process of ADC delivery to the

Figure 12. Selected examples of potent auristatins with sterically demanding C-termini. GI50 values were generated using 786-O renal cell carcinoma cells (20 and 21) and HT29wt colon carcinoma cells (22).55

Figure 13. Representative example of a hydrazone linked auristatin.

The resulting cBR69 (specific to Lewis Y on carcinomas) hydrazone conjugate (DAR = 8) showed good efficacy at 3 mg/ kg in a L2987 human lung adenocarcinoma model. The GI50 value for ADC 23 was determined to be ∼15 ng/mL.27 However, it was also demonstrated that the released ketoester further hydrolyzes to yield the more potent auristatin-E (5, Figure 2) by a second hydrolysis step. The stability of the hydrazone bond in 23 was investigated and half-lives were determined to be 4.4 h (pH = 5) and 183 h (pH = 7). However, upon comparison to the corresponding vc-MMAE ADC (DAR = 8) with N-terminal linker connection (Figure 3), the hydrazone conjugate 23 was less selective in tumor cell F


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Figure 14. Cys-mc-released species of selected auristatin analogues with long intracellular residence times as measured by apparent cell membrane permeabilities [Papp (B > A)] using the Caco-2 flux assay. In comparison, MMAE has a higher recorded value of 73 nm/s. Indicated on the right are data for selected examples of prepared ADCs including ADC GI50 and T/C (tumor vs control) values.

Figure 15. Selected examples of auristatins prepared by Lerchen et al. containing N-terminal carboxylic acid extensions and C-terminal modifications. GI50 values were generated with HT29wt carcinoma cells. The Caco-2 flux assay was used to determine the apparent permeability Papp from B > A.

second bystander mechanism is only observed under physiologically relevant conditions with cleavable linkers and usually does not take place in regular 2D cell assays. This observation indicates that significant deviations from the expected ADC mode of action can occur when switching from a 2D cell based assay format to in vivo conditions, taking under consideration the unique nature of the tumor microenvironment, which can be rich of secreted proteases.59 These

targeted cell. As a consequence, the adjacent tumor and stroma cells are killed in a collateral- and receptor-independent fashion. This is a significant advantage to combat tumor heterogeneity but also has the potential for increased toxicity. We recently demonstrated that there also exists a second mechanism for the bystander effect, which includes the extracellular linker cleavage of ADCs possessing proteolytically cleavable linkers, a mechanism that does not require ADC internalization.59 This G


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Molecular Pharmaceutics findings, which can have a profound impact on ADC design and on the selection of linker-payloads, will be published in a future publication. Regardless of bystander mechanism type, the diffusion potential of the released auristatin species, which is generally a function of the physicochemical properties, needs to be optimized to maximize the bystander effect. Compounds 24−26 (Figure 14) retain high tubulin binding affinity, as determined by the tubulin polymerization assay. Conjugation of the corresponding maleimide linker payloads of 24−26, next to several other structurally related auristatin analogues, to anti-EGFR and C4.4a antibodies provided ADCs with excellent in vitro potencies.57 Several in vivo models were investigated, including the NCI-H292 lung cancer model, and good efficacy was achieved at doses ranging from 2 to 30 mg/ kg. The maleimide analogues of 24−26 and other related analogues were also successfully conjugated to antimesothelin, anti-FGFR2, and anti-CAIX antibodies, providing potent and in vivo-efficacious ADCs.57,60−62 The same authors also investigated several other auristatin analogues having a carboxylic acid substituent on the N-terminus with various new C-termini (Figure 15).55,63,64 Inspection of the permeability values of the compounds shown in Figure 15 reveals that the B > A permeability values vary and do not solely depend on the presence of a negatively charged carboxylic acid function. Notably, epimers 29 and 30 are equipotent, which is another example of how structural changes in the auristatin C-terminal binding domain can be tolerated, in particular regarding stereochemical inversions in certain but not all cases. This observation is corroborated by earlier findings by Bai and Pettit et al., who showed that inversion of the dolaphenine chiral center (from S to R) did not severely compromise potency.65 Dolastatin 10 (1) itself has also been the basis for development of analogues containing hydrophilic N-terminal extensions (Figure 16).66

received considerably less attention. Additionally, most reported work to date has left the isopropyl side chain of dolavaline unchanged, quite possibly a result of the perception that N-terminal modifications are generally less tolerated than C-terminus modifications. For example, work by Miyazaki et al. demonstrated that replacing dolavaline in dolastatin 10 (1) by N,N-dimethyl glycine or N,N-dimethyl alanine has a profound influence on in vivo efficacy in xenograft models, with the glycine analogue demonstrating improved efficacy over dolastatin 10 (1), while the alanine derivative proved nearly inactive.13 In addition, changing the N,N-dimethyl group of dolavaline to a primary amine (valine) significantly reduced in vivo efficacy. While this observation appears to indicate the sensitive nature of the N-terminus with respect to structural modifications, interpretation of in vivo efficacy results in the absence of any data regarding cell-based potency, target engagement, or in vitro ADME, and pharmacokinetics only provides limited value. One example is comparison of the biological properties of dolastatin 10 (1) and symplostatin 1 (37), a naturally occurring analogue of dolastatin 10 (1). The inspection of the cell based cytotoxicity potentials of both analogues indicates that N-terminal modifications on the dolastatin N-termini do not necessarily lead to inactive compounds (Figure 17).3,68

Figure 16. Dolastatin 10 analogues with hydrophilic N-terminal extensions.

In this work, Satomaa et al. demonstrated that propargyl alcohol and azide derivatives 35 and 36, respectively, retaining an appreciable amount of cell potency despite the presence of the hydrophilic side chains. The azide and alkyne functions were employed in subsequent cyclization reactions to form more complex hydrophilic “glycolinkers”, enabling conjugation to various antibodies (lysine, cysteine, glycan, and transglutaminase conjugations). The glycoside linker, which undergoes sialidase-mediated catabolism in the lysosomes after ADC internalization, is functionally similar to peptide linkers and generates the hydrophilic payload derivative inside the cell. Trastuzumab and EGFR1 ADCs were prepared and exhibited good in vivo efficacy at 1.5 to 10 mg/kg (DAR = 3).66,67 2.3. N-Terminal Modifications. In contrast to the many studies that have focused on making structural changes on the auristatin C-terminus, the N-terminal end of the auristatins has

Figure 17. Chemical structures of dolastatin 10 (1) and symplostatin 1 (37). Both naturally occurring peptides only differ at the N-terminal amino acid. Naturally occurring and related analogues symplostatin 3 (38), dolastatin H (39), and isodolastatin H (40) are also shown for comparison. H


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Despite this unknown, a first set of compounds was prepared to specifically interrogate the influence of the N-terminal αcarbon substitution pattern on cell-based activities (Figure 19).

In vitro cytotoxicity of symplostatin 1 (37) against KB cells (GI50 0.15−0.20 nM) and LoVo cells (GI50 0.34−0.50 nM) revealed that 37 is a very potent cytotoxin, with only marginally reduced potency compared to dolastatin 10 (1). The effects of 1 and 37 were examined in A-10 cells by indirect immunofluorescence. Both drugs are potent microtubule depolymerizers. At a concentration of 0.75 nM, dolastatin 10 (1) caused the total loss of cellular microtubules, while a 2.5 nM concentration of symplostatin 1 (37) was required for the same effect.3 Of note are the naturally occurring depsipeptide analogues of dolastatin 10 (1), namely, symplostatin 3 (38), dolastatin H (39), and isodolastatin H (40) (Figure 17).69,70 “Symplostatin 2” has an entirely different structure and is related to dolastatin 13 (not shown).71 Other N-terminal modifications were described by Pettit et al., namely, auristatins with Cbz-protected lysine and arginine as dolavaline replacements, but their cell-potencies were significantly reduced.72 N-Terminal auristatin modifications have also been pursued by Doronina et al., who reported use of p-aminobenzoic acid as the N-terminal amino acid replacement (Figure 18).73

Figure 19. Dolastatin-10 (1) analogues with N-terminal modifications. Activities are expressed as GI50 values obtained with N87 gastric carcinoma cells. For additional cell line data see ref 8.

Auristatin analogues 9 and 46, having the unnatural α,αdimethyl substitution (2-aminoisobutyric acid, Aib), remained highly active with little differentiation from MMAD (6). Interestingly, the potency of 9 and 46 is not dependent on whether or not the terminal amino group is methylated. This stands in contrast to the corresponding N-terminal valine matched pair 6 and 43, where the absence of the N-methyl group in 43 leads to a significant reduction in potency. Also of note are the high cell potencies observed for the glycine and alanine analogues 44 and 45, respectively. These findings indicate that such structural modifications on the N-terminal carbon center are compatible with the auristatin binding mode in the N-terminal tubulin binding cleft. This was also confirmed by crystal structure analyses of 9 and related analogues, which, for the first time, allowed the determination of the detailed auristatin binding mode across the entire peptide.8 Surprisingly, it was observed that the amide bond present between Val and Dil in the tubulin bound structure of 9 adopts the cisconfiguration, whereas all other amide bonds are trans. This result was confirmed by three other auristatin analogues that were also analyzed by tubulin cocrystal structure analysis. However, this data stands in contrast to the observed unbound solution state structures of 9 and dolastatin 10 (1), where the Val-Dil amide bonds are exclusively in the trans-configuration, and only the amide bonds present between Dap and Dil adopt a mixture of both cis and trans isomers at room temperature (Figure 20).8,75

Figure 18. p-Aminobenzoic acid at the auristatin N-terminus: 41, free payload; 42, cAC10 conjugate (DAR = 4). Red, payload; blue, vclinker.

The resulting auristatin 41 showed good cell potency and the cAC10 conjugate 42 led to tumor volume reduction in the Karpas 299 human ALCL xenograft model at 1 mg/kg. However, 42 was found to be less efficacious than the related vc-MMAE ADC.73 More recently, we have reported dolastatin 10 (1) analogues with new N-terminal amino acids bearing tertiary α-carbon centers.8 The design hypothesis for these analogues were based on the published soblidotin crystal structure (2, Figure 2), which reveals a significant binding void present at the Nterminal auristatin binding cleft.9 This observation suggested that additional small substituents attached on the dolavaline αcarbon center should be tolerated. However, at the onset of the studies, it was unclear whether or not the overall binding conformation of the auristatins would be perturbed by such an approach, particularly due to the fact that peptides with α,αdialkyl amino acids can lead to distortions of the peptide by introducing helical turns due to constricted conformational space.74

Figure 20. Cis/trans amide bonds of the Val-Dil-Dap triad in solution at room temperature and in solid state bound to α,β-tubulin. I


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have advanced into phase 1 clinical trials. ADCs employing PF06380101 are targeting main stream solid tumor indications including triple negative breast cancer and nonsmall cell lung c a n c e r , a m o n g o t h e r s ( i . e . , N C T 0 2 12 9 2 0 5 a n d NCT02222922). 2.4. Central Peptide Modifications. In addition to C- and N-terminal modifications on the auristatin peptide (sections 2.1, 2.2, and 2.3) a few published studies also describe modifications to the central peptide core. Gajula et al. reported analogues with mannose- and glucose-derived sugar amino acids as replacements for the dolaproine (Dap) portion of the auristatin peptide (Figure 22).78,79 The rationale for selecting a

Figure 21 shows additional analogues containing N-terminus tertiary α-carbon stereocenters, which were prepared by

Figure 21. Dolastatin-10 (1) analogues containing a tertiary α-carbon center on the N-terminal amino acid. Individual stereoisomers of 50, 51, and 52 were prepared separately and exhibited similar potencies. Activities are expressed as GI50 values obtained with N87 gastric carcinoma cells. For additional cell line data see ref 8.

structure-based drug design. Of these compounds, the high potencies of both pyrrolidine 50 and piperidine 52 analogues are noteworthy.8 Auristatins with cyclic N-terminal motifs, such as the pyrrolidine present in 50, were shown to retain the critical receptor interactions that were identified at the α,β-tubulin interface, which are important for high affinity binding of auristatins. These include a critical hydrogen bond network with Asp β197 located on the tubulin-T5 loop, the amide carbonyl of Phe α351, and a bifocal interaction of the N-2 valine with Asn α329.8 Overall, all analogues containing Nterminal tertiary α-carbon centers were observed to have significantly higher apparent intrinsic clearance values in human liver microsomes and hepatocytes compared to MMAD (6) and MMAE (7), which could lead to lower systemic plasma exposure of free payload after ADC dosing.8 Because of the high cytotoxicity of the auristatins, one desirable goal of developing antibody drug conjugates is to minimize systemic payload exposure in the plasma after ADC dosing, and auristatins with rapid clearance from systemic circulation could lead to a significant safety advantage. It was previously reported that MMAE (7) is mainly cleared by liver oxidative metabolism mediated by CYP3A4, and clinical studies showed that codosing of brentuximab vedotin with the CYP P450 inducer rifampin led to significantly reduced plasma concentrations of unconjugated MMAE payload. 76 This data establishes a direct correlation between the intrinsic clearance capacity of the CYP P450 enzymes and clinical exposure levels of MMAE (7). Even though the systemic plasma payload exposure after ADC dosing is generally very low compared to the corresponding ADC, the absolute exposure values of the unconjugated payload can nevertheless significantly exceed its GI50 values in cell viability assays, even when corrected for plasma protein binding. Hence, the contribution of free payload to general toxicity cannot be underestimated and is a true concern. Attachment of various linkers to the new auristatins containing N-terminal tertiary α-carbon centers provided highly efficacious ADCs on multiple platforms,37,77 and ADCs with the dolastatin 10 analogue 9 (PF-06380101, Figures 4 and 19)

Figure 22. New structural analogues utilizing sugar amino acids. Activities are expressed as GI50 values obtained with HeLa cells.

sugar amino acid was based on the conformational studies of various sugar amino acid oligomers and sugar amino acid-based peptides. Preliminary conformational analysis of analogues such as 53−58 suggested that the dihedral angles of the sugarcontaining amino acids in these molecules would render the backbone structures into a confirmation closely resembling that of the dolaproine (Dap)-containing dolastatin 10 (1). Compound 57 was further examined in cell-based assays with respect to tubulin interference and cell cycle dynamics. Considering the substantial changes made to the overall auristatin structure, the observed cell potency is noteworthy, albeit the activities in cell-based cytotoxicity assays are an order of magnitude lower than those observed for more conventional auristatins. An interesting hybrid approach for development of auristatin analogues was pursued by Zask et al., who combined the structural element of taltobulin (59), a synthetic analogue of the naturally occurring tripeptide Hemiasterlin, with the Cterminal Doe-Dap motif of dolastatin 10 (1) (Figure 23).80 The resulting analogue 60 (Figure 23) is a potent tubulin binding agent and is thought to bind near the vinca binding site in close proximity to the N-terminal auristatin binding cleft.81,82 J


Review


in 59, which has the potential to compromise cell-membrane permeability. In comparison, the hybrid compound 60 shows reduced activity in the same tubulin polymerization assay (62% inhibition), but the cell potency is not significantly different to that of taltobulin (59).80 An important point is that even if the cell-membrane permeabilities were comparable between the compounds, higher potency in the tubulin polymerization assay does not necessarily translate into higher cytotoxicity. This specific disconnect between the cell-free tubulin polymerization assay and cell-based activities in proliferation assays has been known for some time for tubulin inhibitors and was studied in detail by Pettit et al. while examining the potency of dolastatin 10 (1) fragments.65,83 Finally, central peptide modifications were recently described by Park et al., following pursuit of specific variations to the proline peptide region of the auristatins, as exemplified with compounds 61−69 (Figure 24).84 Several potent analogues were prepared with hydroxyl-, methoxy-, and amino-substituents on the pyrrolidine, with one derivative containing a thiazolidine heterocycle 69. These findings indicate that structural changes on the auristatin Dap motif do not significantly compromise potency, but instead represent a promising starting point for future SAR studies. Of particular interest are compounds 67 and 68 because the amine functionality present on the pyrrolidine could also potentially serve as a handle for linker attachment.

Figure 23. Hybrid structure based on taltobulin (59) and dolastatin 10 (1). GI50 values were obtained using KB-3-1 cells.

Similarly, hybrid compound 60 shows subnanomolar potencies in tumor cell proliferation assays but is about an order of magnitude less potent than dolastatin 10 (1). The authors also investigated the Pgp-efflux liabilities of dolastatin 10 (1), 59, and 60, discovering that all three analogues are Pgp efflux substrates with some marginal differences.80 Interestingly, in the cell free tubulin polymerization assay, 59 was found to be more potent than dolastatin 10 (1) (88% inhibition of tubulin polymerization at 0.3 μM vs 78% inhibition for dolastatin 10 (1)), but this difference did not translate into increased cellbased potency. One reason for this discrepancy could be the presence of the negatively charged carboxylic acid functionality

3. CONCLUSION AND FUTURE DIRECTION Since the discovery of dolastatin 10 (1) by Pettit et al. nearly 30 years ago, this class of compounds has drawn widespread interest by a large number of research groups. Such interest has resulted in investigation of many structural changes with respect to modulation of biological properties. Dolastatin 10

Figure 24. Auristatins with proline modifications. GI50 values were obtained with BT474 breast carcinoma cells. K


Review


more auristatin analogues will soon follow. The robust translation for the auristatin-based ADCs from cell-based assays to in vivo and clinical studies is a significant advantage of this platform. Even though payloads with other MOAs and celltargeting profiles are being actively developed, it can be expected that auristatins will remain an important cornerstone for both current and future ADC development.

(1) is one of the most potent tubulin polymerization inhibitors known today, allowing its cytotoxic potential to significantly surpass the activity of most other tubulin-interfering agents. Strong antivascular effects, in addition to its known antimitotic potential, render dolastatin 10 (1) and related synthetic auristatin analogues highly valuable agents for cancer therapy. However, after initial exploration, the excitement about the clinical potential of dolastatin 10 (1) diminished when clinical trials revealed high toxicities at dose levels that did not lead to therapeutically efficacious plasma concentrations. Following this initial setback, several other auristatin analogues with attenuated potency and increased solubility profiles, including auristatin-PE (2, soblidotin, TZT-1027) and auristatin-PYE (3), were developed and advanced into clinical trials. Once again, the clinical outcome for both auristatin derivatives was equally disappointing, as no appreciable therapeutic index could be established. Later, the remarkable potential of this compound class was once again illuminated by the pioneering work of Senter et al. at Seattle Genetics, who discovered that powerful tubulin inhibitors of this type could be very effectively employed as payloads in ADCs. The success stories of brentuximab vedotin (SGN-35) and the many other promising preclinical/clinical ADC projects currently ongoing at Seattle Genetics and their partners were the inspiration for many other academic and industrial research groups to refocus their efforts on auristatins, specifically with the goal of discovering new structural features with the potential for altered biological properties. One example of such an approach was demonstrated by Lerchen et al. (Bayer HealthCare), who aimed to increase the intracellular residence time of auristatin analogues after ADC delivery through attachment of additional Nterminal extensions with carboxylic acid functions (sections 2.2 and 2.3). This strategy is promising for tumor indications in which ADCs with low cell-permeable payloads are effective. However, other tumor indications, i.e., those with low antigen expression across the malignant cell pool, will likely require employment of the bystander effect, which entails the diffusion of the released payload into the extracellular space of the tumor interstitium.29,58,59 The reported tubulin-bound cocrystal crystal structures are of great value for the design of new analogues, providing insights into the detailed auristatin binding mode at the α,β-tubulin interface.8 This includes the surprising result that the Val-Dil amide bond is in cis-configuration in the tubulin bound state. These and other findings will support the structure-based drug design efforts of future analogues. Over the past ten years, many excellent studies about auristatin modifications have been published, and there is a great potential to advance second- and third-generation auristatins as ADC payloads into the clinic. In this regard, an important criterion will be to advance differentiated auristatins having different physicochemical and biological properties, such as increased plasma clearance profiles. Parameters such as payload diffusion potentials, target engagement, and residence times, as well as systemic clearance and distribution profiles will all be related to the auristatin physicochemical properties, and their careful modulation by structure-based drug design will influence the efficacy and safety of future ADCs. At present, all auristatin-based ADCs in clinical trials either employ MMAE (7) or MMAF (8) (Seattle Genetics) as the payloads, with the exception being ADCs with the newly developed dolastatin 10 (1) analogue 9 (PF-06380101, Pfizer) that recently entered clinical trials (Figure 4). Considering the many structural modifications that have been reported, it can be expected that

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AUTHOR INFORMATION

Corresponding Author

*E-mail: andreas.maderna@pfizer.com. Phone: +1-860-715 6498. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS

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REFERENCES

ADC antibody drug conjugate; GI50 50% growth inhibitory concentration; MMAD monomethyl auristatin-D; MMAE monomethyl auristatin-E; MMAF monomethyl auristatin-F; Cys cysteine; DAR drug to antibody ratio; Vc mc-ValCit-PABC (maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl; PABA p-aminobenzyl alcohol; mc maleimidocaproyl; T-DM1 ado-trastuzumab emtansine; AA amino acid; OMe O-methyl; Met methionine; Lys lysine; Asn asparagine; Val valine; Phe phenylalanine; Asp aspartate; Dov dolavaline; Dil dolaisoleuine; Dap dolaproine; Doe dolaphenine; Papp apparent permeability; T/C tumor over control

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