Pyrrolobenzodiazepine Dimer Antibody-Drug Conjugates: Synthesis and Evaluation of Noncleavable Drug-Linkers

Article abstract of DOI:10.1021/acs.jmedchem.7b00736

Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been synthesized via intermediate 19 for use in antibody-drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (6), triazole (7), or piperazine (8) link to the PBD. In vitro IC₅₀ values were 11-48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and 0.10-1.73 μg/mL (7 inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5-1 mg/kg, 1 mg/kg, and 3-6 mg/kg for 6, 8, and 7, respectively. Tumor stasis at 2 mg/kg was observed for anti-CD22 6 in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models.

Full text of DOI:10.1021/acs.jmedchem.7b00736

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Article
Pyrrolobenzodiazepine Dimer Antibody-Drug Conjugates:
Synthesis and Evaluation of Non-Cleavable Drug-Linkers
Stephen J. Gregson, Luke A. Masterson, Binqing Wei, Thomas H. Pillow, Susan D. Spencer,
Gyoung-Dong Kang, Shang-Fan Yu, Helga Raab, Jeffrey Lau, Guangmin Li, Gail D. Lewis
Phillips, Janet Gunzner-Toste, Brian S. Safina, Rachana Ohri, Martine Darwish, Katherine
R. Kozak, Josefa dela Cruz-Chuh, Andrew Polson, John A. Flygare, and Philip W. Howard
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00736 • Publication Date (Web): 07 Nov 2017
Downloaded from http://pubs.acs.org on November 8, 2017
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Pyrrolobenzodiazepine Dimer Antibody-Drug
Conjugates: Synthesis and Evaluation of Non-
Cleavable Drug-Linkers
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,
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Stephen J. Gregson,* Luke A. Masterson, Binqing Wei, Thomas H. Pillow,* Susan D.
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Spencer, Gyoung-Dong Kang, Shang-Fan Yu, Helga Raab, Jeffrey Lau, Guangmin Li, Gail
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D. Lewis Phillips, Janet Gunzner-Toste, Brian S. Safina, Rachana Ohri, Martine Darwish,
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Katherine R. Kozak, Josefa dela Cruz-Chuh, Andrew Polson, John A. Flygare and Philip W.
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Howard
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Spirogen, QMB Innovation Centre, 42 New Road, London, E1 2AX, United Kingdom
‡
Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States
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ABSTRACT: Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been
synthesized via intermediate 19 for use in antibody-drug conjugates (ADCs). They lack a
cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a
hydrophilic spacer, and either an alkyne (6), triazole (7) or piperazine (8) link to the PBD. In
vitro IC s were 11–48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the anti-HER2
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ADCs (HER2 0 MCF7, all inactive) and 0.10–1.73 µg/mL (7 inactive) in CD22 3+ BJAB and
WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo anti-tumor
efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5–1 mg/kg, 1
mg/kg and 3–6 mg/kg for 6, 8 and 7, respectively. Tumor stasis at 2 mg/kg was observed for
anti-CD22 6 in WSU-DLCL2. In summary, non-cleavable PBD-ADCs exhibit potent activity,
particularly in HER2 models.
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INTRODUCTION
There is a great deal of pharmaceutical industry interest in the development of antibody-drug
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conjugates (ADCs) for the treatment of cancer. The majority of ADCs have a common design
concept which generally consists of three variable parts: a tumor antigen targeting monoclonal
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antibody, a covalent linking group and a cytotoxic warhead. Two ADCs have been on the
market for several years (Figure 1): trastuzumab emtansine (1, HER2 [human epidermal growth
factor receptor 2] targeting, non-cleavable linker, emtansine warhead) and brentuximab vedotin
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2, CD30 targeting, cleavable dipeptide linker, self-immolative p-aminobenzyl [PAB] spacer,
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monomethyl auristatin E warhead).
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At present there are >60 ADCs in clinical trials and several of these contain the pyrrolo[2,1-
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c][1,4]benzodiazepine (PBD) ring system as the cytotoxic component of the ADC (Figure 1).
PBDs were originally discovered in the 1960s and a number of natural products containing the
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ring system were isolated from various Streptomyces species. The mode of anti-tumor action
was later established and shown to involve sequence-selective alkylation in the minor groove of
DNA, where an N2 of guanine forms a covalent bond with the electrophilic N10/C11 imine on
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the PBD. Advances in PBD synthesis enabled the preparation of many synthetic derivatives
including dimeric DNA cross-linking PBDs with extended base sequence recognition capability
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and increased cytotoxic potency. One such PBD dimer, SJG-136, was evaluated as a standalone
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agent in phase I/II clinical trials for cancer.
With the resurgence of interest in ADC technology, it became apparent that potent PBD dimers
were ideal candidates for the cytotoxic component. The number of potential chemical linking
positions in PBDs offers great flexibility for antibody connection and this is apparent in the two
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Figure 1] which feature in
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3 is linked via the C2 position of the PBD
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and 4 via the N10 position and to the antibody through a maleimide-cysteine connection. Both
molecules contain a dipeptide (valine-alanine) trigger which is cleaved by cathepsin in the
lysosome. A self-immolative PAB spacer is necessary in 4 to liberate the bis-imine DNA cross-
linking PBD dimer upon catabolism of the ADC, while the PEG group aids water solubility. An
indolobenzodiazepine (IBD) 5 (IMGN779) is also in clinical development as an ADC drug-
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linker and is linked to the antibody through an aryl tether in the IBD. Again, the linkage in 5 is
cleavable (disulfide trigger) but it is linked to the antibody through a lysine connection.
Given that a non-cleavable linker is employed in the approved drug 1, we wanted to apply this
notion to the PBD dimer platform, and envisaged that linking through an aryl tether would
provide a symmetrical molecule and facilitate the possibility of a non-cleavable linkage to an
antibody. A non-cleavable linker similar to that of 1 could give active metabolites from
lysosomal antibody degradation that may be less membrane permeable and decrease potential
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toxicities in neighboring healthy tissue through lack of bystander effect. Another motive for
investigating non-cleavable PBD-ADCs was that although dipeptide cleavable linkers are stable
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in human and primate plasma models, some are unstable in rodent plasma. Therefore,
premature drug release prior to internalization may affect efficacy and tolerability data when
assessing cleavable ADCs in rodents at the preclinical stage.
As proof of principle for the novel non-cleavable ADCs, HER2 (found in many solid tumors)
and CD22 (found in many hematological cancers) were attractive antigen targets to investigate.
There are several next generation anti-HER2 ADCs in development including topoisomerase 1
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inhibitor-based and duocarmycin-based payloads, and a biparatopic ADC containing a
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tubulysin warhead . For CD22, inotuzumab ozogamicin has demonstrated promising clinical
activity and has recently received FDA approval for patients with relapsed or refractory acute
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lymphoblastic leukemia. We wish to report molecular modelling, synthesis, and efficacy
evaluation of the first examples of non-cleavable anti-HER2 (trastuzumab, 4D5) and anti-CD22
PBD ADCs based on three drug-linker molecules 6, 7 and 8 (Figure 2). The compounds
described here consisted of either an alkyne (6), triazole (7) or piperazine (8) incorporated in the
PBD aryl tether and a maleimide connecting group for site-specific antibody cysteine
conjugation. A PEG spacer was included in the linkers to enhance conjugation solubility in
aqueous media. These molecules were accessible from the key iodoaryl intermediate 19 using
either Sonogashira, click or Buchwald chemistries.
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The belief was the active drugs released from ADCs of 6, 7 and 8 would be cysteine adducts
and this hypothesis was based on published evidence for other non-cleavable ADCs: In the case
of marketed non-cleavable ADC 1, catabolism in the lysosome yields the released drug as a
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lysine adduct of the cytotoxin emtansine,
similarly the non-cleavable ADC cAC10-
maleimidocaproyl-monomethylauristatin F releases a cysteine adduct of monomethylauristatin
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Figure 1. Structures of marketed ADC drug-linkers trastuzumab emtansine (1) and brentuximab
vedotin (2), a standalone PBD dimer and PBD (3 and 4)/IBD (5) ADC drug-linkers in clinical
trials and development. Notable PBD atom numbers are shown in 3.
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Figure 2. PBD dimer ADCs evaluated in this study. The drugs were conjugated to either anti-
HER2 (thio Hu anti-HER2 4D5 HC-A118C) or anti-CD22 (thio Hu anti-CD22 10F4v3 HC-
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RESULTS
Molecular Modelling: Previous studies have shown that PBD dimer 4a, containing a
pentyldioxy tether, efficiently forms DNA interstrand cross-links in a sequence-dependent
fashion, and exhibited sub-nanomolar anti-proliferative activities against a panel of cancer cell
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lines (Figure 3a).
Based on the solution structure of a PBD monomer covalently bound in a
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standard B-DNA helix (PDB entry 2KTT ), we modelled how PBD dimers, including 4a, would
bind in a 10-nucleotide long DNA duplex (5’-AACCATCGTT-3'). In a similar fashion to the
pentyl tether in 4a, modelling predicted that released linker-cysteine adducts containing a 1,3-
bismethyl benzene tether would fit equally as well into the DNA minor groove (Figure 3b).
Furthermore, modelling suggested that substitution at the 5-position of the benzene tether could
directly protrude out of the minor groove without interfering with the DNA backbone, and
provide a novel way of linking a symmetrical PBD dimer to the antibody. Candidate non-
cleavable drug-linkers were evaluated in terms of their steric and electrostatic interactions
complementary to the minor groove of DNA (particularly the phosphate backbone), and their
conformational preferences. The search identified three diverse scaffolds that contain an alkyne,
a triazole, or a piperazine moiety, respectively (6, 7 and 8 in Figure 2). For example, in the
model of compound 6 bound to DNA (Figures 3c and 3d), two covalent bonds (bond length 1.47
Å~1.49 Å) between the C11/C11’ of the PBD and the exocyclic N2 atoms of two
deoxyguanines, separated by 4 base pairs on opposing DNA strands, serve to anchor the PBD
dimer snugly inside the minor groove. Potential hydrogen bonds between the N10 of the PBD
and an adjacent DNA base may also contribute to binding. Alkyne substitution on the central
benzene allows a tether linker to reach above the phosphate rim of the DNA minor groove. The
rest of the linker provides ample separation between the antibody attachment point (maleimide-
cysteine, Figure 2) and the DNA minor groove. Assuming the ethylene glycol moieties adopt an
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energetically favored gauche conformation, the separation was estimated to be >10 Å. For
drug-linker compounds 7 and 8, the triazole and piperazine moieties were predicted to be largely
co-planar with the benzene tether due to conjugation, and were well accommodated in the DNA
minor groove (Figures 3e and 3f). A similar separation of the DNA and the antibody point for
that observed with 6 was also realized for 7 and 8.
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Figure 3. (a) Structure of pentyldioxy tethered PBD dimer 4a. (b) Schematic representation of an
aryl tether PBD dimer bound to 10-nucleotide long DNA duplex (5’-AACCATCGTT-3'). (c)
Model of 6 (carbon atoms in cyan) bound in the DNA minor groove, the molecular surface of the
drug-linker cysteine adduct is shown in yellow. (d) Alternative view of the same complex with 6
looking down the minor groove. The covalent bonds between C11/C11’ of the PBD and the
exocyclic N2 atom of two deoxyguanines are marked by solid lines and labeled with the bond
length values (in Å), and potential hydrogen bonds between N10/N10’ of the PBD and an
adjacent DNA base are marked by dashed lines. (e) Model of 7 (carbon atoms in brown) bound
in the DNA minor groove. (f) Model of 8 (carbon atoms in green) bound in the DNA minor
groove.
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Chemistry: The known nitrobenzoic acid 9 was converted to its corresponding acid chloride
and coupled to methyl (2S,4R)-4-hydroxypyrrolidine-2-carboxylate.HCl to give amide 10 in 68%
yield (Scheme 1). The methyl ester was reduced to the diol 11 in 90% yield, followed by SnCl2
mediated reduction of the nitro group. The crude aniline 12 was Boc protected to give 13 in 60%
yield, which was followed by simultaneous ring closure and secondary alcohol oxidation to
provide ketone 14. Debenzylation by hydrogenolysis, followed by protection of the free phenol
and C11-OH with THP, gave 16 which was isolated as a mixture of diastereoisomers arising
from the THP group. Wittig olefination followed by selective cleavage of THP from the phenol
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furnished 18, in which the C11 THP group was still required to prevent racemisation at C11a
during the subsequent dimerization step. A DMF solution of known 1,3-bis(bromomethyl)-5-
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iodobenzene (obtained in 2 steps by LiBH reduction then Appel halogenation from
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commercially available dimethyl 5-iodoisophthalate) was reacted at 60 °C with two equivalents
of phenol 18 in the presence of K CO and TBAI to provide the key iodoaryl intermediate 19 in
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The synthesis of alkyne drug-linker 6 from intermediate 19 is shown in Scheme 2. Initially, a
linear approach was explored starting with a Sonogashira reaction on 19 with propargylamine,
followed by amide coupling with 1-maleimido-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-
oic acid. This was unsatisfactory due to the instability of the resulting aryl propargylamine 22a
affording small quantities of 24 in low overall yield (Scheme 2 and Figure 4). Instead, a more
convergent approach was adopted, which involved reacting propargylamine with 2,2-dimethyl-4-
oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-oic acid (20) to give the Sonogashira coupling
partner 21 in 71% yield. The subsequent Sonogashira reaction on 19 was performed under
microwave conditions and gave 22 in 70% yield. Attempted triple Boc deprotection of 22 to give
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3b with 95% TFA/H O was unsuccessful and instead resulted in polymerization using these
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conditions. Therefore, selective primary N-Boc deprotection was investigated as an alternative
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approach. This involved a two stage process where the Boc carbamate was converted to a TBS
carbamate followed by TBAF cleavage to reveal the desired amine. Although the Boc removal
was not as selective as anticipated – side products 23a and 23b resulting from secondary N-Boc
cleavage were observed by LC-MS – recovery was respectable and crude material was carried
through to the next step. Amine 23 (and side products 23a and 23b) was coupled to N-maleoyl-β-
alanine to provide a mixture of predominantly 24, plus 24a and 6. Treatment with 95% TFA
afforded drug-linker 6 in 60% yield over the 4 steps.
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0
1
2
3
4
5
6
7
8
9
0
The synthesis of triazole-linked drug-linker 7 is shown in Scheme 3. Alkyne 25 was obtained
in 95% yield using Sonogashira on 19 under microwave conditions at 100 °C, and subsequent
TMS cleavage with K CO gave click substrate 26 in 78% yield. Copper-catalyzed click reaction
2
3
of 26 with 11-azido-3,6,9-trioxaundecan-1-amine provided triazole 27. Amide coupling of 27
with 6-maleimidohexanoic acid N-hydroxysuccinimide ester was slow and gave precursor 28 in
4
5% yield. Cleavage of Boc and THP protecting groups with 95% TFA furnished 7 in 53% yield.
4
1
Buchwald chemistry was chosen for installation of the piperazine in drug-linker 8; a solution
of 19 in the presence of base, Boc-protected piperazine and RuPhos/RuPhos Pd G1 methyl t-
butyl ether adduct was heated at 85 °C in a sealed tube to give 29 in 34% yield (Scheme 4). The
formation of unwanted de-halogenated 19 was minimized by using cesium carbonate as base
rather than sodium t-butoxide. Simultaneous THP cleavage and triple Boc deprotection with 95%
TFA gave 30, which was carried through without purification. Finally, EDCI coupling of 1-
maleimido-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oic acid with 30 gave 8 in 32% yield.
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Page 13 of 67
Journal of Medicinal Chemistry
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7
8
9
a
Scheme 1. Synthesis of 19
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Reagents and conditions: (a) 1. (COCl) , DCM, DMF, RT, 16 h; 2. methyl (2S,4R)-4-
2
hydroxypyrrolidine-2-carboxylate.HCl, DCM, TEA, -20 °C, 16 h, 68% yield; (b) LiBH , THF, 0
C, 4 h, 90% yield; (c) SnCl , MeOH, reflux, 3 h, 12 carried through crude; (d) (Boc) O, THF,
reflux, 16 h, 60% yield; (e) TEMPO, DAIB, DCM, RT, 16 h, 46% yield; (f) 10% w/w Pd-C,
EtOH, RT, 4 h, 53% yield; (g) DHP, PTSA, EtOAc, RT, 1 h, 65% yield; (h) PPh CH Br, KO Bu,
THF, 0 °C to RT, 18 h, 72% yield; (i) THF, AcOH, H O, RT, 3 h, 87% yield; (j) 1,3-
bis(bromomethyl)-5-iodobenzene, K CO , TBAI, DMF, 60 °C, 3 h, 91% yield.
4
°
2
2
t
3
3
2
2
3
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Journal of Medicinal Chemistry
Page 14 of 67
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 2. Synthesis of 6
H
O
O
O
N
H
a
O
O
Boc
b
O
O
O
N
HN
O
O
Boc
OH
20
21
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
H
N
H
N
O
O
O
O
O
R
O
O
O
N
O
O
HN
HN
O
O
Boc
N
Boc
N
THPO
H
OTHP
H
Boc
N
Boc
THPO
OTHP
H
O
OMe
O
MeO
d
O
O
N
H
N
N
N
OMe
MeO
N
O
O
O
24
O
2
2: R = Boc
c
23: R = H
O
H
N
O
O
O
N
O
O
HN
O
O
e
N
O
OMe
O
N
H
H
N
MeO
N
6
O
O
a
Reagents and conditions: (a) Propargylamine, EDCI, DCM, RT, 16 h, 71% yield; (b) 19,
Pd(PPh ) , CuI, diethylamine, DMF, 4Å molecular sieves, microwave at 100 °C, 26 min, 70%
yield; (c) 1. TBSOTf, 2,6-lutidine, DCM, RT, 16 h; 2. TBAF, THF, RT, 1 h, 23 carried through
crude; (d) EDCI, N-maleoyl-β-alanine, DCM, RT, 3 h, 24 carried through crude; (e) 95%
TFA/H O, 0 °C, 1 h, 60% yield from 22 (in 4 steps).
3
4
2
Figure 4. Structures of compounds to assist discussion of Scheme 2.
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Page 15 of 67
Journal of Medicinal Chemistry
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7
8
9
a
Scheme 3. Synthesis of 7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Reagents and conditions: (a) TMS-acetylene, Pd(PPh ) , CuI, diethylamine, DMF, microwave
3
4
at 100 °C, 30 min, 95% yield; (b) K CO , MeOH, RT, 3 h, 78% yield; (c) 11-azido-3,6,9-
trioxaundecan-1-amine, CuSO .5H O, sodium ascorbate, BuOH, H O, RT, 16 h, 27 carried
through crude; (d) 6-maleimidohexanoic acid N-hydroxysuccinimide ester, DCM, RT, 3 d, 45%
yield; (e) 95% TFA/H O, 0 °C, 1 h, 53% yield.
2
3
t
4
2
2
2
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Journal of Medicinal Chemistry
Page 16 of 67
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 4. Synthesis of 8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) tert-butyl-piperazine-1-carboxylate, RuPhos Pd G1 methyl t-
butyl ether adduct, RuPhos, Cs CO , THF, 85 °C, 4 h, 34% yield; (b) 95% TFA/H O, 0 °C, 30
min, 30 carried through crude; (c) EDCI, 1-maleimido-3-oxo-7,10,13,16-tetraoxa-4-
azanonadecan-19-oic acid, DCM, RT, 2 h, 32% yield.
2
3
2
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Page 17 of 67
Journal of Medicinal Chemistry
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9
Antibody Conjugation: The maleimide PBD drug-linkers 6, 7 and 8 were conjugated to
mutated anti-HER2 and anti-CD22 antibodies at position HC-A118C. Site specific cysteine
engineered antibodies were chosen which contain engineered cysteines to give a theoretical
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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56
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59
60
42
drug:antibody ratio (DAR) of 2 for each ADC. Average DAR, aggregation analysis and
percentage remaining free drug for the 6 ADCs are shown in Table 1. In summary, the
maleimides were reacted in 6–8-fold molar excess with de-blocked, re-oxidized, cysteine
engineered antibodies. The crude ADCs were then purified by cation exchange chromatography
and characterized by UV spectroscopy to determine protein concentration. Analytical size-
exclusion chromatography (SEC) was used for aggregation analysis and LC-MS determined
DAR.
Table 1. Analysis of ADCs
a
d
e
PBD
mAb
DAR
1.9
% Monomer
95.6
% Free drug
b
6
anti-HER2
<0.5
c
anti-CD22
1.7
1.8
1.8
1.8
1.8
98.6
95.6
95.0
95.6
94.2
b
<0.5
<0.5
<0.5
<1.0
<3.0
b
7
8
a
anti-HER2
c
anti-CD22
b
anti-HER2
c
anti-CD22
DAR = drug:antibody ratio determined by LC-MS. Thio Hu anti-HER2 4D5 HC-A118C.
Thio Hu anti-CD22 10F4v3 HC-A118C. Determined by SEC. Determined by LC-MS.
c
d
e
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Journal of Medicinal Chemistry
Page 18 of 67
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In Vitro Cytotoxicity. The anti-HER2 ADCs were evaluated for their in vitro cytotoxicity in 3
breast cancer cell lines with differing levels of HER2 expression based on
immunohistochemistry: MCF7 (HER2 0), KPL-4 (HER2 3+) and SK-BR-3 (HER2 3+). As
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
28, 43
anticipated, the anti-HER2 ADCs displayed no activity in the HER2 0 MCF7 negative
control cell line (Figure 5c, Table 2). 8 and 6 both showed good potency in HER2 3+ KPL-4
with IC values of 28 ng/mL and 48 ng/mL, respectively; 7 was inactive in KPL-4 (Figure 5b).
5
0
All 3 anti-HER2 ADCs showed potent activity in HER2 3+ SK-BR-3 line with IC values of 11,
5
0
15 and 23 ng/mL for 6, 8 and 7 (Figure 5a), respectively. The control anti-CD22 ADC for 8 was
inactive across all lines (Figure 5a–c).
Similarly, the anti-CD22 ADCs were tested in 2 CD22 3+ lymphatic lines (BJAB and WSU-
DLCL2) and 1 CD22 0 hematological line (Jurkat). The anti-CD22 ADCs and anti-HER2
controls were inactive at low doses in the CD22 0 line Jurkat, which was as expected (Figure 6c,
Table 3). Anti-CD22 6 and 8 gave IC s of 0.10 and 0.18 µg/mL, respectively, in BJAB cells and
50
1.22 and 1.73 µg/mL, respectively, in WSU-DLCL2 cells (Figure 6a–b). Anti-CD22 7 and the
control anti-HER2 ADCs did not show any activity in the CD22 3+ lines.
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Page 19 of 67
Journal of Medicinal Chemistry
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Table 2. In Vitro Cytotoxicity Data in HER2 Models
PBD
mAb
IC (ng/mL)
50
c
d
d
MCF7
KPL-4
SK-BR-3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
a
a
6
7
8
8
anti-HER2
anti-HER2
anti-HER2
>10,000
>10,000
>10,000
>10,000
48.25
11.14
22.90
15.00
>10,000
28.20
b
anti-CD22
>10,000
>10,000
a
b
c
Thio Hu anti-HER2 4D5 HC-A118C. Thio Hu anti-CD22 10F4v3 HC-A118C. HER2 0.
HER2 3+.
d
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Journal of Medicinal Chemistry
Page 20 of 67
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9
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1
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1
1
1
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3
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5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. In vitro cytotoxicity data for the PBD ADCs in HER2 models (a) SK-BR-3 (b) KPL-4
and (c) MCF7.
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Page 21 of 67
Journal of Medicinal Chemistry
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7
8
9
Table 3. In Vitro Cytotoxicity Data in CD22 Models
PBD
mAb
IC (µg/mL)
50
c
d
d
Jurkat
WSU-DLCL2
BJAB
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
a
6
7
8
6
7
8
anti-CD22
anti-CD22
>20
>20
>20
>20
>20
>20
1.22
>20
1.73
>20
>20
>20
0.10
>20
0.18
>20
>20
>20
a
anti-CD222
b
anti-HER2
b
anti-HER2
b
anti-HER2
a
b
c
Thio Hu anti-CD22 10F4v3 HC-A118CThio. Hu anti-HER2 4D5 HC-A118C. CD22 0.
CD22 3+.
d
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Journal of Medicinal Chemistry
Page 22 of 67
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7
8
9
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1
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2
2
2
2
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3
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3
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6
0
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2
3
4
5
6
7
8
9
0
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2
3
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. In vitro cytotoxicity data for the PBD ADCs in CD22 models (a) BJAB (b) WSU-
DLCL2 and (c) Jurkat.
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Page 23 of 67
Journal of Medicinal Chemistry
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9
In Vivo Efficacy. Investigation of the in vivo single-dose efficacy for the 3 non-cleavable
PBDs was performed in the Founder 5 (Fo5) mammary tumor transplant model (Figure 7a–c,
Table 4) for the anti-HER2 conjugates, and the WSU-DLCL2 lymphoma xenograft model
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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48
49
50
51
52
53
54
55
56
57
58
59
60
(
Figure 7d, Table 4) for the anti-CD22 ADCs. The anti-CD22 ADCs acted as controls in the Fo5
experiments and similarly the anti-HER2 compounds were used as negative controls for WSU-
DLCL2. The 3 anti-HER2 ADCs showed good efficacy in the Fo5 model which was dose-
responsive. Anti-HER2 6 and 8 were tested at 0.3, 1 and 3 mg/kg and the doses to give tumor
stasis were 0.3–1 mg/kg and 1 mg/kg, respectively (Figure 7a–b). In the case of 6 and 8, the
control anti-CD22 ADCs at 3 mg/kg did show activity at a similar level to anti-HER2 6 at 0.3
mg/kg and anti-HER2 8 at 1 mg/kg. From the in vitro experiments, anti-HER2 7 was less active,
so was tested at 3 and 6 mg/kg in Fo5 and the dose to give tumor stasis was 3–6 mg/kg, however,
there was more of a differential from the control ADC at 6 mg/kg (Figure 7c). Anti-CD22 6 was
tested at 0.5 and 2 mg/kg in WSU-DLCL2 and showed good efficacy at the higher dose level
versus the anti-HER2 control at 2 mg/kg (Figure 7d). Anti-CD22 7 did not show any activity at 5
mg/kg, similarly anti-CD22 8 did not show efficacy at 0.5 mg/kg in WSU-DLCL2.
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Journal of Medicinal Chemistry
Page 24 of 67
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4. In Vivo Efficacy Summary
PBD
Tumor stasis dose (mg/kg)
a
b
Fo5
WSU-DLCL2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
7
8
a
0.3–1
2
3–6
>5
>0.5
1
b
Thio Hu anti-HER2 4D5 HC-A118C. Thio Hu anti-CD22 10F4v3 HC-A118C.
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Page 25 of 67
Journal of Medicinal Chemistry
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48
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52
53
54
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56
57
58
59
60
Figure 7. In vivo efficacy data for the PBD ADCs, single dose at day 0: (a) 6 (b) 8 and (c) 7 in
the Fo5 model and (d) 6, 7 and 8 in the WSU-DLCL2 model.
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Journal of Medicinal Chemistry
Page 26 of 67
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1
1
1
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1
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1
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2
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3
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4
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5
5
5
5
5
5
5
5
6
CONCLUSION
Non-cleavable linkers in ADCs are known to change the active metabolite and thus can greatly
influence the safety and efficacy profiles for this class of drugs. As the parameters that make up a
successful ADC are currently not well-understood, and are dependent on the antigen target and
disease indication, the largest possible tool box of linker-types for a given drug class is of great
value.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Six examples of PBD dimer ADCs possessing non-cleavable linkers were designed and
successfully synthesized. Molecular modelling studies, with a 10-nucleotide long DNA duplex
(5’-AACCATCGTT-3'), suggest that linking the antibodies through aryl tethers would not have a
detrimental effect on the ability of the released PBD cysteine catabolites to bind in the minor
groove of DNA. In vitro cytotoxicity evaluation showed the anti-HER2 PBD-ADCs to have
potent activity in the HER2 3+ cell line SKBR-3 (IC s 11–23 ng/mL), however, a slight
5
0
reduction in activity in HER2 3+ KPL-4 (IC 28–48 ng/mL, 7 inactive) was observed. Complete
5
0
abolition of cytotoxicity in the HER2 0 MCF7 line demonstrated selectivity in vitro with these
ADCs for higher HER2-expressing cell lines. Similar results were obtained in the CD22 in vitro
models for anti-CD22 6 and 8 with IC s of 0.10–0.18 µg/mL in BJAB CD22 3+ cells and 1.22–
50
1.73 µg/mL in WSU-DLCL2 CD22 3+ cells, while no activity was observed in the CD22 0
Jurkat line for anti-CD22 6, 7 and 8 at low doses. Anti-CD22 7 had poor activity against CD22
3+ expressing lines. For both HER2 and CD22 models, negative control ADCs did not display
activity in the 3+ expressing cell lines.
The anti-HER2 ADCs of the 3 compounds showed promising in vivo anti-tumor efficacy in the
HER2-expressing Fo5 breast model. The rank order of potency from the in vitro studies was
reflected in vivo with HER2 conjugates of 6 and 8 requiring similar doses to reach tumor stasis
(0.3–1 mg/kg for 6 and 1 mg/kg for 8), with 7 being less active (3–6 mg/kg). In vivo activity in
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the Fo5 model with the control anti-CD22 ADCs was more pronounced with 6 and 8 than 7,
which could simply be due to 7 being less efficacious. However, it should be noted that the
targeted anti-HER2 ADC of 6 was active at about 1/10 the dose of control and in the case of 8
about 1/3 the dose of control. In the CD22 model WSU-DLCL2, only anti-CD22 6 showed
activity (2 mg/kg) compared to negative control HER2 (2 mg/kg) at the doses tested.
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Overall, the ADCs performed better in HER2 targeted models than CD22 in vitro and in vivo;
for both targets ADCs derived from alkyne 6 and piperazine 8 were more potent than triazole 7.
Given the modelling suggests steric factors would be similar between the drug-linkers, we
wondered whether polarity may explain the difference in activity for 7. The calculated
(MarvinSketch 15.1.12.0) LogDs at pH 7.4 of the three drug-linkers were 3.49 (7), 1.72 (6) and
1.48 (8), which suggests 7 would penetrate cell membranes better than 6 and 8 and thus be more
potent, however, the reverse was observed with 6 and 8 being the most active. The multifactorial
nature of ADC constructs (e.g. linker type, polarity, warhead potency, internalization potential,
ability to reach drug target etc.) make it difficult to discriminate in terms of biological activity
between three drug-linker molecules which broadly come in the same class. Structure activity
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relationships of PBDs have determined how to fine tune PBD warhead activity, but the effects
of the linking group from the aryl tether of a PBD-ADC on biological activity are not clearly
understood at the present time.
From a chemistry perspective, the syntheses of 7 and 8 were facile from the key iodoaryl
intermediate 19. The synthesis of 6 was the most challenging and although the methodology was
not ideal (unwanted partial secondary N-Boc cleavage), the recovery of material for antibody
conjugation was respectable. Iodoaryl intermediates of type 19 are versatile and could be useful
for the introduction of antibody linking groups using other palladium chemistries.
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The data presented here support the molecular modelling hypothesis that non-cleavable linking
of an ADC through the aryl tether of a PBD dimer does not interfere with PBD-DNA binding
and biological activity. Mode of action studies on this class of non-cleavable PBD-ADCs are
underway and the results will be published elsewhere. Two PBD drug-linker molecules
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containing dipeptide cleavable linkers are currently being evaluated in the clinical setting, the
work described here suggests that non-cleavable PBD dimer ADCs show potential and warrant
further evaluation for PBD-ADC tool box inclusion.
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EXPERIMENTAL SECTION
Molecular Modelling. Modelling was based on the solution structure of a PBD monomer
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covalently bound to a 10mer B-DNA duplex (PDB entry 2KTT ). The DNA sequence was
mutated to 5’-AACCATCGTT-3' to introduce two dG nucleotides at an appropriate
configuration for alkylation with the PBD dimers. The C11(S) geometry was adopted for all
PBD molecules studied, as this stereochemistry is known to lead to energetically favored adducts
with the exocyclic C2-NH2 group of an embedded guanine in the context of a B-DNA duplex.
The length of the covalent bond between the C11 of the PBD and the exocylic amino N atom of
deoxyguanine was constrained to a value between 1.47 Å and 1.49 Å, consistent with the
observations from PBD structure 2KTT. Local minimizations of the complex including the
above constraints were performed in MOE (version 2015, Chemical Computing Group) using the
default parameters. The models were visually examined and manual adjustments were made as
needed in MOE to correct some artifacts of the local minimization.
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Synthesis of Compounds. Fine chemicals and reaction solvents were purchased from Sigma-
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Aldrich and chromatography solvents were bought from Fisher Scientific. MAL-dPeg -CO H
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(1-maleimido-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oic acid) and t-Boc-N-amido-
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dPEG ₄-acid (20, 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-oic acid) were
obtained from Quanta BioDesign, Ltd. Optical rotations were measured on an ADP 220
polarimeter (Bellingham Stanley Ltd.) and concentrations (c) are given in g/100 mL. IR spectra
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were recorded on a Bruker Alpha Platinum-ATR. H and C NMR spectra were acquired at 300
K using a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts
are reported relative to TMS (δ = 0.0 ppm), and signals are designated as s (singlet), d (doublet),
t (triplet), dd (doublet of doublets) or m (multiplet), with coupling constants given in Hertz (Hz).
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