Natural Product Splicing Inhibitors: A New Class of AntibodyâDrug

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Natural Product Splicing inhibitors: A New Class of Antibody-Drug Conjugate (ADC) Payloads Sujiet Puthenveetil, Frank Loganzo, Haiyin He, Ken Dirico, Michael E. Green, Jesse A. Teske, Sylvia Musto, Tracey Clark, Brian Rago, Frank E. Koehn, Robert Veneziale, Hadi Falahaptisheh, Xiaogang Han, Frank Barletta, Judy Lucas, Chakrapani Subramanyam, Christopher J. O'Donnell, L. Nathan Tumey, Puja Sapra, Hans-Peter Gerber, Dangshe Ma, and Edmund Idris Graziani Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00291 • Publication Date (Web): 14 Jul 2016 Downloaded from http://pubs.acs.org on July 14, 2016

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Bioconjugate Chemistry

Natural Product Splicing inhibitors: A New Class of Antibody-Drug Conjugate (ADC) Payloads Puthenveetil, Sujiet1, Loganzo, Frank2, He, Haiyin1, Dirico, Ken1, Green, Michael1, Teske, Jesse1,5, Musto, Sylvia2, Clark, Tracey3, Rago, Brian3, Koehn, Frank1, Veneziale, Robert4,6, Falahaptisheh, Hadi4, Han, Xiaogang3, Barletta, Frank3, Lucas, Judy2, Subramanyam, Chakrapani1, O’Donnell, Christopher J.1, Tumey, L. Nathan1, Sapra, Puja2, Gerber, Hans Peter2, Ma, Dangshe2,7, and Graziani, Edmund I.1* 1

Oncology Medicinal Chemistry, Pfizer, 445 Eastern Point Rd., Groton CT 06340

2

Oncology-Rinat Research & Development, Pfizer, 401 N. Middletown Rd., Pearl River, NY 10965

3

Pharmacokinetics, Dynamics and Metabolism, Pfizer, 445 Eastern Point Rd., Groton CT

4

Drug Safety Research and Development, Pfizer, 401 N. Middletown Rd., Pearl River, NY 10977

5

current address: AbbVie Inc., 1 North Waukegan Road, North Chicago, Illinois 60064, United States

6

current address: Global Drug Safety Research and Evaluation (DSRE), Takeda California, 10410 Science Center Drive, San Diego, CA 92121

7

current address: Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591

* Corresponding author, [email protected] Tel. (860)-715-6768

ACS Paragon Plus Environment


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Abstract There is a considerable ongoing work to identify new cytotoxic payloads that are appropriate for antibody-based delivery, acting via mechanisms beyond DNA damage and microtubule disruption, highlighting their importance to the field of cancer therapeutics. New modes of action will allow a more diverse set of tumor types to be targeted and will allow for possible mechanisms to evade the drug resistance that will invariably develop to existing payloads. Spliceosome inhibitors are known to be potent anti-proliferative agents capable of targeting both actively dividing and quiescent cells. A series of thailanstatin-antibody conjugates were prepared in order to evaluate their potential utility in the treatment of cancer. After exploring a variety of linkers, we found that the most potent antibody-drug conjugates (ADCs) were derived from direct conjugation of the carboxylic acid-containing payload to surface lysines of the antibody (a “linker-less” conjugate). Activity of these lysine conjugates was correlated to drug-loading, a feature not typically observed for other payload classes. The thailanstatinconjugates were potent in high target expressing cells, including multi-drug resistant lines, and inactive in non-target expressing cells. Moreover, these ADCs were shown to promote altered splicing products in N87 cells in vitro, consistent with their putative mechanism of action. In addition, the exposure of the ADCs was sufficient to result in excellent potency in a gastric cancer xenograft model at doses as low as 1.5 mg/kg that was superior to the clinically approved ADC T-DM1. The results presented herein therefore open the door to further exploring splicing inhibition as a potential new mode-of-action for novel ADCs.

Introduction Antibody drug conjugates (ADCs) are a promising class of cancer therapeutics that employ the specificity of an internalizing monoclonal antibody (mAb) to deliver a covalently attached cytotoxic small molecule payload to tumor cells that overexpress the target antigen as compared to normal cells.1 Conceptually, the judicious choice of antigen combined with the stability of the conjugate in circulation result in reduced adverse effects as compared to highly potent small molecule cytotoxic agents. This improvement in safety is largely driven by reduced exposure of the free cytotoxic agent in healthy tissue. Recently approved ADCs have employed microtubule inhibitors (MTIs) such as monomethylauristatin E (MMAE)2 and maytansines3 in order to induce cell cycle arrest and subsequent cell death via destabilization of tubulin subunits. These agents are very effective against rapidly dividing cancer cells; however there is a need to identify other classes of ADC payloads that are effective against


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both rapidly and slowly proliferating cells.4 This need has recently spurred a renewed interest in RNA polymerase inhibitors5 and DNA damaging agents6, 7 as ADC payloads.

A number of groups recently identified a series of analogs8, 9 of the natural product FR901464 (1), including spliceostatin C (2), and thailanstatin A (3), that are ultra-potent inhibitors of eukaryotic RNA splicing. (Figure 1) These compounds bind tightly to the SF3b subunit of the U2 snRNA sub-complex, an essential component of the spliceosome.10, 11 Manipulation of the biosynthetic genes that control production of these analogs in the bacteria have allowed for sufficient material to be generated for a medicinal chemistry approach to be used in evaluating this mechanistic class as potential payloads for next generation ADCs.12, 13 We herein report the identification of a new natural product ADC payload that has been used to generate potent and efficacious ADCs. The ADCs can specifically kill cells that express both high and low levels of antigen, but do not target cells that are antigen negative. In contrast to microtubule inhibitors such as monomethylauristatin E (MMAE) and maytansines, these spliceostatin ADCs are able to overcome a multi-drug resistant (MDR) phenotype.

Figure 1: Structure of FR901464 (1), spliceostatin C (2), and thailanstatin A (3)

Results

Maleimide Containing Linkers Bearing Spliceostatin Analogs Failed to Conjugate Due to an Intermolecular Diels-Alder Reaction: Thailanstatin A (3) was derivatized to the linker-payload (4) bearing an amide spacer with a maleimide conjugation handle using standard amide coupling conditions (Scheme 1A). When 4 was reacted with trastuzumab in an attempt to make a conventional cysteine conjugate, the reaction failed to produce conjugate 5. Careful mass spectral and NMR analysis revealed that compound 4 had undergone an intramolecular Diels-Alder reaction to give the cyclic derivative 6, thus rendering it devoid of a conjugation handle. The intramolecular Diels-Alder reaction could be avoided by shortening the linker length or by using a rigid diamine spacer such as 1,4-diaminocyclohexane in place of the



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ethylenediamine spacer and these LPs could be successfully conjugated to trastuzumab via a conventional cysteine conjugate. However, the redesigned linker-payloads were found to exhibit limited chemical stability due to the undesired slow intermolecular Diels-Alder reaction. Due to these complexities, maleimide-based conjugation approaches were deprioritized.

Haloacetamide and NHS-esters Yield Successful Cysteine and Lysine Conjugates of Thailanstatin: In an effort to circumvent the chemical stability challenges with maleimide based linkers attached to this payload two approaches were explored to generate ADCs of thailanstatin A. In an initial approach, a haloacetamide based cysteine conjugation handle was prepared. Thus, thailanstatin A (3) was derivatized to the linker-payload (7) bearing an ethylenediamine haloacetamide linker by coupling to Fmoc protected ethylene diamine followed by removal of the Fmoc group and acetylation of the resulting amine with iodoacetic acid (Scheme 1B). Next, the intrachain disulfide bonds of trastuzumab were reduced with TCEP and the resulting free cysteine thiol groups were reacted with 7 to give a heterogeneous conjugate 8 with an average drug-antibody ratio (DAR) of 6.2.

Alternatively, thailanstatin A (3) was activated as its N-hydroxysuccinimide (NHS) ester (9) and was conjugated to the lysines on trastuzumab yielding “linker-less” ADCs. These direct conjugates were prepared by addition of 3-12 equivalents of 9 to trastuzumab in 50 mM borate buffer (pH 8.7) at room temperature. Purification via size exclusion chromatography (SEC) resulted in ADCs (10a-e) with different average drug-antibody ratios, depending upon the equivalents of payload used (10a: DAR ≤ 1.6; 10b: DAR=1.6-2.5; 10c: DAR = 2.5-3.5; 10d: DAR = 3.5-4.5; 10e: DAR > 4.5). DAR was estimated by LCMS under reducing conditions by quantitation of the +518 signals on both the light chain and heavy chain. Additional MS analysis following tryptic digestion confirmed the thailanstatins to be conjugated across multiple lysines on both the light and heavy chains.

A: Maleimide-linked conjugates to hinge-cysteines


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B: Iodoacetamide-linked conjugates to hinge-cysteines

C: Amide-linked conjugates to lysines

Scheme 1. Preparation of various thailanstatin conjugates. Conditions: a) HATU, TEA, DMF; b) 6maleimidopentanoic acid, DCC, DMF c) trastuzumab, PBS, TCEP (2 eq), 1-3h, rt; d) HATU, TEA, DMF, temp; e) i) 9H-fluoren-9-ylmethyl (2-aminoethyl)carbamate , Hunigs base, DMF, temp; ii) piperidine, temp; f) iodoacetic acid, DCC, DMF, temp; g) trastuzumab, PBS, TCEP (5 eq), 1.5h, 37°C; h) N-hydroxyl succinimide, DCC, THF, 0 ˚C; i) trastuzumab, 9 (3-12 eq), 50 mM borate, pH 8.7.

ADCs Bearing Splicing Inhibitor Payloads Exhibit Potent Cytotoxicity and Selectivity for Antigen Positive Cancer Cell Lines: The cytotoxicity of trastuzumab-thailanstatin ADCs was assessed by a plate-based assay using cell line models with varying levels of Her2 expression. In cell lines with high Her2 expression (“3+”), corresponding to >600,000 receptors per cell (i.e. N87, BT474, HCC1954), the potency of ADCs 10a to 10e was relatively independent of drug-antibody ratio (DAR). For example, for N87 and BT474 cells the IC50 ranged from approximately 13 to 43 ng/ml, with no apparent dependence on DAR (Table 1 and Supplemental Figure S2). For HCC1954, the IC50 was less than 173 ng/ml, with minimal DAR dependence. In contrast, for cell lines expressing moderate levels of Her2 (“2+”), in the range of ~70 – 170,000 receptors per cell (i.e. MDA-MB-DYT2, MDA-MB-453, JIMT1), the drug loading of trastuzumab-



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thailanstatin ADCs had a dramatic effect on potency (Table 1 and Supplemental Figure S2). In MDA-MB361-DYT2 cells, for example, the IC50 decreased to approximately 77 ng/ml only when DAR was 3.5 or greater (ADCs 10d and 10e) while low DAR conjugates were approximately 200-800 fold less potent. In JIMT1 cells, which are inherently less sensitive to T-DM1 and other trastuzumab ADCs, thailanstatin A drug loading greater than 4.5 was necessary to achieve potency below 300 ng/ml. The lower loaded ADCs were >300 fold less potent. In contrast to the ADCs in series 10, trastuzumab-thailanstatin ADC 8 was ineffective in cell lines with moderate antigen levels despite having a loading of ~6. In MDA-MB361-DYT2 and JIMT1 cells, the IC50 was >23,800 ng/ml while in higher expressing cells lines (N87 and BT474) the IC50 was 28 ng/mL and 84 ng/mL, respectively. Each of these ADCs showed minimal cytotoxicity in Her2-negative cells, as evidenced by the high IC50’s in MDA-MB-468 cells (>24,000 ng/ml). These data suggest that antigen expression level and the drug loading may greatly impact the efficacy of trastuzumab-thailanstatin ADCs.

Table 1: In vitro cytotoxicity results for ADCs IC50 (ng/ml antibody) Her2

+++

+++

+++

+++

++

++

++

-

N87

N87-

BT474

HCC1954

MDA-MB-

MDA-MB-

JIMT1

MDA-MB-

361-DYT2

453

level ADC

DAR

MDR1

468

8

6.3

28

nd

84

30

>23,800

nd

>23,800

>23,800

10a

≤ 1.6

20

116

39

48

60,406

nd

>125,000

>125,000

10b

>1.6 to

25

46

42

47

11,577

1812

>68,000

>68,000

25

63

43

52

1448

262

>460,000

>460,000

26

nd

36

172

77

78

13,750

>36,000

2.5 10c

>2.5 to 3.5

10d

>3.5 to 4.5

10e

>4.5

13

nd

29

36

25

78

246

>24,000

11

2.2

64

nd

36

242

>30,000

>26,000

>30,000

>30,000

T-DM1

3.5 – 4.2

62

>60,000

190

16

40

38

1073

3140

Data are reported as Mean IC50 antibody ng/ml. Antigen expression levels are designated: +++ as ~500K - 1M per cell, and ++ as ~60K - 175K per cell, and negative as 900-fold.

Antigen expression level impacts ADC potency: In order to explicitly test the effect of antigen level on potency of selected trastuzumab-thailanstatin A ADCs, MDA-MB-361-DYT2 cells (“2+”) were transduced with additional Her2 protein. Immunoblot confirmed that the resultant 361-Her2 cells had higher (“3+”) levels of Her2 protein, comparable to N87 gastric cancer cells (Figure 2A). Cytotoxicity assessment demonstrated that the potency of ADC 10b (DAR 2.2) increases when higher levels of Her2 are expressed in the same cell background (Figure 2B). The IC50 for 10b was determined to be approximately 3940 ng/ml in parental 361 cells as compared to 18 ng/mL in 361-Her2 cells, an increase in potency of >130-fold. This activity in 361-Her2 cells was equivalent to the IC50 observed in N87 cells (18 ng/mL) that have endogenously high expression of Her2. Hence, antigen level impacts the potency of low DAR trastuzumab-thailanstatin A ADCs.



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Figure 2: Induction of Her2 levels increases potency of low DAR trastuzumab-thailanstatin ADC. (A) Her2 levels in retrovirally-transduced 361-Her2 cells compared with parental 361 and high Her2 expressing N87 cells. (B) MDA-MB-361-DYT2 (361) parental, 361-Her2 transduced, and N87 cells were treated with the indicated concentration of ADC 10b and assessed for cytotoxicity. The potency of 10b increases in 361-Her2 compared with parental 361 cells.

Thailanstatin A-ADCs alter splicing of protein products: Unconjugated thailanstatin A analogs were previously shown to upregulate the cyclin-dependent kinase inhibitor protein, p27, and to produce altered splice products (p27*) in several cell lines10. We confirmed a similar observation using a trastuzumab-thailanstatin ADC. When N87 gastric cancer cells were treated for 48h with ADC 10b, levels of full length p27 and truncated p27* increased in a dosedependent fashion (Figure 3). These data confirm that delivery of thailanstatin A payload via an antibody can modulate RNA splicing.

Figure 3: Spliceostatin ADC alters splicing of p27. Gastric cancer N87 cells were treated with trastuzumab-thailanstatin ADC 10b (DAR 2.2) at 0, 1, 3, and 9 nM payload concentration (corresponding


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to 0, 68, 205, and 614 ng/mL ADC) for 48h. Probing of cellular protein with anti-p27 antibody detected increased levels of full length p27 and truncated p27* protein products in the presence of ADC 10b.

Pharmacokinetic Parameters in Mouse for ADCs 10b-d: Plasma samples were collected at 0.083, 6, 24, 48, 96, 168, and 336 hours after a 1mg/kg IV dose administration of ADCs 10b, 10c, and 10d to nu/nu (nude) mice. Concentrations of total Ab (measurement of both conjugated and unconjugated mAb) or ADC (mAb conjugated to 1 or more drug molecules) were determined using a ligand binding assay (Figure 4). Circulating ADC was captured using immobilized sheep anti-human IgG. The total antibody (tAb) and ADC exposure was determined using a goat anti-human IgG and rabbit anti-payload polyclonal reagent, respectively. Total Ab exposure for all 3 ADCs (t1/2 = 122-161h) was in the expected range and showed a slight DAR-dependence, consistent with previous literature reports.14, 15 However, ADC exposure was unexpectedly low for all 3 samples, exhibiting only 25-30% of the tAb exposure (t1/2 = 79-91h). These results suggested that the circulating ADC was either losing payload (by deconjugation- presumably via a retro-Michael mechanism) or the payload was being metabolized over time to a form not recognized by the anti-payload reagent.

Immuno-capture high resolution LCMS was used to characterize the in vivo DAR over time (Figure 5C) and qualitatively asses any metabolism of linker or payload. ADC was first deglycosylated (PNGase F), and then captured from blood samples using immobilized anti-human IgG. LCMS analysis of the intact ADC allowed for DAR determination and samples were reduced with TCEP for metabolite ID. (Figure 5A) This analysis on the reduced ADC showed a loss of 42 Daltons suggesting the acetate group of the thailanstatin payload was being hydrolyzed over time, resulting in increasingly heterogeneous ADC. (Figure 5B) Synthesis of this metabolite (compound 11) and evaluation of its in vitro cytotoxicity showed that loss of the acetate did not have a significant impact on ADC potency (vide infra). Therefore, this metabolite was included as an active species in the DAR calculations. Counting both acetyl and desacetyl ADC as active, it was clear that the ADC was more stable in circulation than the LBA-based method suggested. (Figure 5C) Loss of DAR by LCMS occurred at approximately the same rate in both rat and mouse.



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Figure 4: Pharmacokinetics of thailanstatin ADCs in mice. Concentration vs time profiles following a 1 mg/kg IV dose administration of the various thailanstatin ADCs in nu/nu mice. Concentrations of total Ab (measurement of both conjugated and unconjugated mAb) or ADC (mAb conjugated to 1 or more drug molecules) were determined using a ligand binding assay. Profiles were used for calculation of mouse pharmacokinetic parameters.


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Figure 5: Metabolism of thailanstatin ADCs to active metabolite, 11. (A) Immuno-capture LCMS under reducing conditions in nu/nu mice following 1 mg/kg administration, zoomed in on the LC+1 peak revealing the loss of 42 Daltons taking place over time; (B) Proposed active metabolite (11) of thailanstatin ADC 10; (C) In vivo change in DAR following a 3 mg/kg dose administration to Sprague Dawley rat and 1 mg/kg dose in nu/nu mice as measured by high resolution LCMS, including species 10 and 11.



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ADCs Bearing Splicing Inhibitor Payloads Are Efficacious in Her2 Positive Xenograft Mice Models of Cancer The in vivo efficacy of trastuzumab thailanstatin A conjugates 10b-10d (DAR = 2.2, 3.2, and 4.2, respectively) was evaluated in a N87 gastric cancer xenograft model. Animals were randomized and the tumors were allowed to grow until they reached approximately 350 mm3, at which point dosing was initiated intravenously at 0.5, 1.56, and 3 mg/kg. ADCs were dosed on days 0, 4, 8, and 12 (q4d x 4). Dose-dependent responses were observed for all three ADCs (Figure 6A-7C). Nearly complete regression was observed at the highest dose tested (3 mg/kg) and 4-5 mice (out of 8) from each ADC tested had no measureable tumor at experimental termination (day 97). However, the 0.5 mg/kg dose did not provide any significant therapeutic benefit for any of the ADCs. Differences between the efficacy of the 3 ADCs was most pronounced at the mid-dose (1.56 mg/kg) as shown in Figure 6D where the DAR 3.2 ADC (10c) is superior to the DAR 2.2 and DAR 4.2 ADCs (10b and 10d). All three compounds were found to be considerably more efficacious than the clinically approved ADC T-DM1 (Supplemental Figure S1).


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Figure 6: Thailanstatin ADCs are efficacious in N87 gastric cancer models. (A, B, C) Conjugates 10b (DAR2.2), 10c (DAR 3.2), and 10d (DAR 4.2), respectively, exhibit dose-dependent efficacy and result in nearly complete tumor regression when dosed at 3 mg/kg (mpk); (D) Comparison of amide conjugates 10b, 10c, and 10d at 1.56 mg/kg.

Discussion Antibody drug conjugates bearing microtubule inhibitors (MTIs) as payloads have demonstrated clinical efficacy against a number of tumor types, but there remain many tumors that either do not respond to MTI-based ADCs or develop resistance to them. This is particularly true of slow growing tumor cells which are typically not as dependent upon tubulin-mediated cell processes as are rapidly dividing cell types. Highly potent compounds that induce cell death via a non-tubulin mechanism of action are therefore attractive as payloads for ADCs that may kill both dividing and quiescent cells. A number of specific mechanisms focusing on DNA damaging agents to induce cell death have been and are being evaluated for ADCs both clinically and pre-clinically. These include double strand DNA cleavage via the natural product calicheamicin,16 DNA intercalation via anthracylines17 such as doxorubicin, as well as DNA alkylation via duocarmycins18 and pyrrolobenzenodiazepine dimers.19

With the aim of expanding the scope of potent ADC cytotoxins beyond MTIs and DNA damaging agents, we turned our attention to inhibitors of RNA splicing. mRNA processing is a critical step in translation; newly synthesized pre-mRNA is further edited and assembled by a very large protein complex termed the spliceosome. Natural product inhibitors of mRNA splicing, spliceostatin C (2) and thailanstatin A (3), exert their effects via non-covalent binding to the SF3b subunit of the U2 snRNA subcomplex of the spliceosome and show low-nM to sub-nM IC50 values against multiple cancer cell lines. Additionally, a semisynthetic analog of pladienolide D, E7107, is being evaluated in human clinical trials for cancer,20 providing early clinical support for employing spliceosome inhibitors as ADC payloads.

We recently reported8 an evaluation of semi-synthetic analogs of spliceostatin and thailanstatin and showed that semisynthetic ester and amide analogs of 2 and 3 exhibited significantly higher potency against tumor cell lines than the corresponding free acids, presumably due to increased permeability. The observation that amides prepared at the carboxy terminus of the compound maintained good activity suggested to us that attachment of a linker at this position might not interfere with binding to the spliceosome. We therefore initially employed a strategy to prepare spliceostatin-based linker-



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payloads (LPs) whereby an amide analog of 2 and 3 is attached to an ADC via a non-cleavable linker. Trastuzumab was employed as a model antibody due to its well-studied biophysical properties and proven clinical utility. The high expression level of its target (Her2) in various tumor cell lines and minimal expression in normal tissue makes it an ideal model system for the study of new cytotoxic modalities.

Early attempts to conjugate maleimide-containing thailanstatin LP 4 to trastuzumab gave surprisingly poor loading. Careful analysis of the linker-payload (LP) stock solution used for conjugation revealed a significant amount of 4 had undergone an intramolecular Diels-Alder cyclization to form product 6. The structure of 6 was confirmed by NMR, and this decomposition pathway is likely the underlying cause of the poor loading. In order to overcome this issue, the reactive electrophile moiety at the conjugation terminus of the linker-payload was modified from a maleimide to a haloacetamide, thus preventing any diene-related reactivity. Haloacetamides typically react slightly more slowly than maleimides with thiols, however they have been reported to yield conjugates of comparable activity and likely improved stability.21 Therefore, linker-payload 7 was prepared and conjugated to the hinge disulfides of trastuzumab yielding the relatively high-loaded ADC 8 (average DAR = 6.2). Gratifyingly, 8 demonstrated potent cytotoxicity in cell lines with high Her2 expression (Table 1) with IC50s in the range of 25-40 ng/mL and minimal cytotoxicity in a non-Her2 expressing cell line (MDA-MB-468). However, in a moderate Her2 expressing cell line, MDA-MB-361-DYT2, ADC 8 had an IC50 of >21000 ng/mL suggesting that this ADC may not be effective against cell types with lower target expression. Encouragingly, these results were the first indication that if sufficient antigen is present on the target cell, an active thailanstatin moiety can be released from the ADC and make its way to the spliceosome target to effect efficient and dose-dependent cell killing.

Given the observed drop-off in in vitro activity in lower antigen expressing cell lines, we went on to explore the hypothesis that the nature of the released species from the ADC may play a role in the activity of the conjugate. In an effort to simplify the linker, and hence release a modified payload closer in structure to the previously reported8 and highly potent amide of thailanstatin, we conjugated an activated ester of the payload, 9, directly to trastuzumab. By varying the stoichiometry and reaction time we were able to produce and isolate various kinetically controlled lysine-linked ADCs having average DARs ranging from approximately 1 to 6, 10a-e. Interestingly, all these ADCs showed potent cytotoxic activity against high Her2 expressing cell lines (N87, BT474, HCC1954, Table 1), having IC50’s


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generally in the range of 13-50 ng/mL. However, in moderate Her2 expressing MDA-MB-361-DYT2 cells, the potency was strongly correlated with the DAR of the conjugate. Low DAR ADCs (3.5) exhibited IC50’s in the range of 25-80 ng/mL. The same trend was observed with two other low-to-moderate expressing cell lines, MDA-MB-453 and JIMT1. (Table 1, Supplemental Figure 2) The latter cell line in particular was only sensitive to ADCs with DAR > 4.5, such that thailanstatin ADCs having a loading (DAR) less than 4 showed IC50s generally >100,000 ug/mL. This super-stoichiometric relationship between loading and IC50 has been previously observed in a series of maytansinoid ADCs.22, 23 However, the DAR-dependence in those studies was much more modest than the present results. Our studies indicate that increasing the DAR of a conjugate 2-fold can increase the potency of the resulting ADC by as much as 500-fold.

The DAR sensitivity of the MDA-MD361-DYT2 cells was shown to be directly dependent on antigen expression. The sensitivity of this cell line to compound 10b (DAR 2.2) could be increased by approximately 2 orders of magnitude by induction of Her2 expression (Figure 2). Together with the DAR-sensitivity data, this is strong evidence that these compounds are essentially saturating the available cell surface antigens, thereby limiting the delivery of payload to its site of action. The amount of payload being delivered can be increased by either increasing the number of payloads delivered per binding event (i.e. increasing the DAR) or by increasing the number of antigens available for ADC binding.

Resistance to various chemotherapeutics is often mediated by overexpression of efflux pumps such as MDR1 (ABCB1, P-glycoprotein).24 In order to study how this phenomenon may impact thailanstatin ADCs, the high-HER2 expressing gastric cancer line N87 was engineered to overexpress MDR1. Consistent with literature reports,25 we found that the clinically approved ADC T-DM1 is approximately 900-fold less potent in this engineered cell line. Encouragingly, both thailanstatin 2 and its corresponding ADCs 10a-10c (Table 1) were found to be quite effective in an MDR1-overexpressing cell line, with potencies approximately equivalent to those in non-engineered cell line. This strongly suggests that the payload and the closely related lysosomally-released species (presumably a lysinelinked thailanstatin) are not MDR1 substrates and thus may be effective in chemotherapy-resistant tumors.



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With a number of potent thailanstatin ADCs now in hand, we proceeded to evaluate the in vivo efficacy of this payload in a representative model of Her2 positive gastric cancer. Given our lack of prior in vivo experience with thailanstatin ADCs, it was necessary to first to determine what doses would be tolerated in rodents. Doses up to 3 mg/kg were found to be well tolerated in both rats and mice, and therefore dosing in the efficacy model was performed at 0.5, 1.56, and 3 mg/kg (see below). Pharmacokinetics in mice (1 mg/kg) and toxicokinetics in rats were also determined in order to evaluate exposure and metabolism in efficacy and toxicity species.

The total antibody exposure in nu/nu (nude) mice was found to have a slight dependence on DAR, similar to previous reports of auristatin-linked ADCs (Figure 4).14, 15 The correlation between antibody clearance and DAR is speculated to be due to higher liver accumulation of hydrophobic (higher DAR) ADC species.26 ADCs 10b-d consist of mixtures of high-DAR and low-DAR species resulting in the average loading shown in Table 1. While the exposure differences between ADCs 10b, 10c, and 10d are subtle, they fit well with these prior reports. More significantly, the ADC concentration as measured by LBA was found to drop off within the first 48 hours of exposure. By 48h, the LBA assay indicated that only about 20% of the ADC remained in circulation. However, concurrent efficacy studies (vide infra) showing that the compounds exhibited excellent tumor regression at comparable doses raised questions about the large difference between tAb and ADC exposure.

In order to further investigate these results, pharmacokinetic and toxicokinetic samples from mice and rats, respectively, were evaluated by immunocapture high resolution LC/MS in order to evaluate the loss of payload (by determining the DAR loss over time) and possible payload metabolism. Reduction and deglycosylation of the samples revealed that a loss of 42 Daltons was taking place over time from the loaded light-chain ADC peak (Figure 5A). Similar changes took place on the heavy chain (data not shown). We propose that this loss of 42 Daltons corresponds with a loss of the acetate from the payload, possibly mediated by a plasma esterase (Figure 5B). Unlike previous reports of esterase mediated cleavage,27 the metabolism of thailanstatin ADCs was found to take place in both mice and rats (data not shown). Compound 11 was synthesized and found to be an active metabolite, having similar in vitro cytotoxicity to parent compound 10 in the N87 cell line. A DAR 2 version of compound 11 was found to have an IC50 of 28 ng/mL against N87 cells but an IC50 of >25000 ng/mL against DYT2 cells. Given the similar potency of 10 and 11 in the high-expressing cells, we chose to treat the desacetyl metabolite as active drug in our DAR calculations. In this revised analysis, we were able to show that


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>75% of the payload was retained over the course of 4 days of exposure. In comparison, the LBA results had suggested significant loss of payload over time (ADC/tAb ratio was 23-31%). LBA data generated with a new anti-payload reagent raised against both the parent acetyl and active desacetyl metabolite correlated well with the DAR results (data now shown) and suggested that the original anti-payload reagent was not binding to the active desacetyl metabolite.

The efficacy of compounds 10b, 10c, and 10d was evaluated in a high-Her2 expressing gastric cancer cell line (N87). The compounds were all generally ineffective at 0.5 mg/kg and highly effective at 3 mg/kg (Figure 6). At the 3mg/kg dose, all three compounds were found to be considerably more efficacious than the clinical comparator compound, T-DM1 (Supplemental Figure 1). The most significant differences in efficacy between the compounds, however, were observed at the mid-dose (1.56 mg/kg). At this dose, the DAR 3 species (10c) significantly outperformed both the lower DAR species (10b) and the higher DAR species (10d) (Figure 6D). Compound 10c also outperformed both the higher and lower DAR species at the high dose (3 mg/kg), although the differences were more subtle. Complete tumor regression was observed in 5 out of 8 mice at the 3 mg/kg dose of compound 10c, compared to 2/8 and 3/8 mice for compounds 10b and 10d, respectively. Given the improved in vitro cytotoxicity of the higher loaded ADCs in various cell lines (vide supra), it was perhaps surprising that the highest DAR ADC (10d) did not provide the most efficacy. The most plausible explanation for this observation is the higher antibody exposure of 10c (1030±158 ug*hr/mL) as compared to 10d (725±172 ug*hr/mL) at the 1 mg/kg dose. Decreased exposure of higher DAR ADCs has also been offered as an explanation for the reduced efficacy observed for DAR6 vcMMAE conjugates as compared to the corresponding DAR4 conjugates.14 Together, these results are a reminder that optimization of ADC efficacy requires a careful balance of drug loading and exposure. This balance between loading and exposure must be uniquely tuned for the specific linker-payload that is being advanced.

Both 10b and 10c were evaluated in single-dose toxicity study in rats at doses up to 10 mg/kg.

A single

IV dose of Compound 10b was well tolerated up to 10mg/kg, and did not demonstrate any severe toxicity or significant changes in body weight. These findings were considered non adverse, therefore, 10 mg/kg was identified as the non-observed adverse effect level (NOAEL). Compound 10c, the most efficacious ADC on a per-dose basis, was found to be well tolerated in rats up to 3 mg/kg without any significant toxicity, a dose that was identified as the NOAEL for this compound. Importantly, both 10b and 10c showed nearly complete N87 tumor regression at doses equal to or less than the NOAEL.



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Conclusions This work discloses the first exploration of RNA splicing inhibitors as payloads for antibody drug conjugates (ADCs), an important emerging and clinically validated therapeutic class for cancer treatment. The paper describes the significant challenges that were overcome in order to design and prepare novel antibody conjugates employing a natural product payload with an orthogonal mechanism of action to anything studied previously. Conjugates prepared exhibit significant potency against nonproliferating slow growing tumor cell lines as well as drug resistant cell lines and demonstrate efficacy in animal models of cancer with a superior profile to T-DM1, an approved ADC in clinical use.

Experimental Procedures: See Supporting information for preparation of payloads, linkers, conjugation conditions, in vitro cell line development, immunoblots, in vivo efficacy, pharmacokinetics and safety studies.

Supporting Information General procedures, methods for compound preparation, methods for preparation of conjugates, methods for cell line development, methods for cytotoxicity assays, methods for immunoblots, methods for in vivo experiments, methods for pharmacokinetic analyses, tables of cytotoxicity, animal body weights, and full PK parameters.

Acknowledgments We thank Xiang Zhang for optimization of conjugations and purifications of spliceostatin ADCs, Ellie Muszynska and Nadira Prashad for expert analytics of various ADCs.

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