Discovery of an SSTR2-Targeting Maytansinoid Conjugate (PEN-221) with Potent Activity in Vitro and in Vivo

Article abstract of DOI:10.1021/acs.jmedchem.8b02036

Somatostatin receptor 2 (SSTR2) is frequently overexpressed on several types of solid tumors, including neuroendocrine tumors and small-cell lung cancer. Peptide agonists of SSTR2 are rapidly internalized upon binding to the receptor and linking a toxic payload to an SSTR2 agonist is a potential method to kill SSTR2-expressing tumor cells. Herein, we describe our efforts towards an efficacious SSTR2-targeting cytotoxic conjugate; examination of different SSTR2-targeting ligands, conjugation sites, and payloads led to the discovery of 22 (PEN-221), a conjugate consisting of microtubule-targeting agent DM1 linked to the C-terminal side chain of Tyr³-octreotate. PEN-221 demonstrates in vitro activity which is both potent (IC₅₀ = 10 nM) and receptor-dependent (IC₅₀ shifts 90-fold upon receptor blockade). PEN-221 targets high levels of DM1 to SSTR2-expressing xenograft tumors, which has led to tumor regressions in several SSTR2-expressing xenograft mouse models. The safety and efficacy of PEN-221 is currently under evaluation in human clinical trials.

Full text of DOI:10.1021/acs.jmedchem.8b02036

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Article
Discovery of an SSTR2-targeting maytansinoid conjugate
(PEN-221) with potent activity in vitro and in vivo
Brian H. White, Kerry Whalen, Kristina Kriksciukaite, Rossitza G. Alargova, Tsun Au Yeung,
Patrick Bazinet, Adam Hale Brockman, Michelle Dupont, Haley Oller, Charles-Andre
Lemelin, Patrick Lim Soo, Benoit Moreau, Samantha Perino, James M. Quinn, Gitanjali
Sharma, Rajesh Shinde, Beata Sweryda-Krawiec, Richard Wooster, and Mark T. Bilodeau
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b02036 • Publication Date (Web): 08 Feb 2019
Downloaded from http://pubs.acs.org on February 9, 2019
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of their duties.

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Discovery of an SSTR2-targeting maytansinoid
conjugate (PEN-221) with potent activity in vitro
and in vivo
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Brian H. White*, Kerry Whalen, Kristina Kriksciukaite, Rossitza Alargova, Tsun
Au Yeung, Patrick Bazinet, Adam Brockman, Michelle DuPont, Haley Oller,
Charles-Andre Lemelin, Patrick Lim Soo, Benoît Moreau, Samantha Perino,
James M. Quinn, Gitanjali Sharma, Rajesh Shinde, Beata Sweryda-Krawiec,
Richard Wooster, Mark T. Bilodeau
Tarveda Therapeutics, 134 Coolidge Ave. Watertown, MA 02472, U.S.A.
Abstract
Somatostatin receptor 2 (SSTR2) is frequently overexpressed on several types of solid
tumors, including neuroendocrine tumors and small cell lung cancer. Peptide agonists of
SSTR2 are rapidly internalized upon binding to the receptor and linking a toxic payload
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to an SSTR2 agonist is a potential method to kill SSTR2-expressing tumor cells. Herein
we describe our efforts towards an efficacious SSTR2-targeting cytotoxic conjugate;
examination of different SSTR2 targeting ligands, conjugation sites, and payloads led to
the discovery of 22 (PEN-221), a conjugate consisting of microtubule-targeting agent
DM1 linked to the C-terminal side chain of Tyr3-octreotate. PEN-221 demonstrates in vitro
activity which is both potent (IC50 = 10 nM) and receptor dependent (IC50 shifts 90-fold
upon receptor blockade). PEN-221 targets high levels of DM1 to SSTR2-expressing
xenograft tumors, which has led to tumor regressions in several SSTR2-expressing
xenograft mouse models. The safety and efficacy of PEN-221 is currently under
evaluation in human clinical trials.
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Introduction
Antibody-drug conjugates (ADCs) have shown promise in improving the therapeutic
index of anti-cancer agents through selective targeting of active agents to tumor cells
while lowering systemic toxicity. The approval of brentuximab vedotin1, trastuzumab
emtansine2 and inotuzumab ozogamicin3 have demonstrated the value of antibody-
directed therapies in the treatment of patients with cancer. However, despite the clinical
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promise ADCs have demonstrated, several challenges preclude their use in the treatment
of patients with solid tumors. Solid tumors are frequently poorly vascularized4 and the rate
of diffusion of antibodies through solid tumor tissue is extremely slow5, which can lead to
low levels of ADC uptake within solid tumors6. One potential alternative approach to
ligand-directed therapies of solid tumors is the use of miniaturized constructs, such as
peptide- or small molecule-drug conjugates7. While small (>5 kDa) constructs typically
suffer from poor pharmacokinetics, their ability to rapidly diffuse through solid tumor tissue
frequently allows for extremely high levels of targeted construct uptake into solid tumors8.
Somatostatin is a peptide hormone produced by paracrine cells within the
gastrointestinal tract, lung, and pancreas, and the central nervous system. Somatostatin
is produced in two active forms (SST14 and SST28), and the biological effects include
neurotransmission, inhibition of gastrointestinal endocrine secretion and cell proliferation.
These effects are produced by somatostatin’s interaction with five somatostatin receptors
(SSTR1-5). While somatostatin itself is cleared within minutes in vivo9, investigations into
peptide analogs of somatostatin have yielded longer-lived peptide analogs selective for
all somatostatin receptor subtypes10. Of particular note is the peptide analog octreotide11,
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a long-lasting SSTR2 agonist that inhibits secretion of growth hormone through its action
at SSTR2 and is approved for use for treatment of acromegaly and symptomatic
treatment of patients with carcinoid tumors.
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In addition to its role in the endocrine system, SSTR2 is overexpressed on the cell
surface of various types of solid tumors, including small-cell lung cancers12, gastroentero-
pancreatic neuroendocrine tumors13, carcinoids and islet cell tumors14. Expression of
SSTR2 is limited outside the central nervous system, and since SSTR2 agonists are
rapidly internalized into SSTR2-expressing cells upon receptor engagement15, SSTR2-
mediated drug targeting represents a promising method to deliver compounds into
SSTR2-expressing tumors16. As part of our ongoing effort to explore miniaturized drug
conjugates that can rapidly target chemotoxic payloads to solid tumors, we set out to
develop an SSTR2-targeted peptide-payload construct that demonstrated SSTR2-
mediated cytotoxicity and was able to induce tumor regressions of several different
SSTR2-expressing xenografts.
Results and Discussion
Scheme 1. Synthesis of payload/linker combinations
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O
O
HN
OH
O
H
HN
OH
O
H
O
O
O
a
O
O
6
7
O
O
O
S
N
N
SH
N
S
O
O
N
N
8
9
O
O
DM1
1
Cl
Cl
O
O
S
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S
N
BzO
O
HO
OAc
BzO
HO
OAc
H
O
OH
H
O
O
O
O
b
O
Ph
O
Ph
O
BocHN
O
BocHN
O
OMe
MeO
OMe
MeO
cabazitaxel
2
O
H
N
N
O
HN
N
H
H
O
OH
O
O
O
N
H
monomethyl auristatin E
c
O
O
O
H
S
N
N
O
N
S
O
N
N
H
H
O
OH
O
O
O
O
O
N
H
3
O
O
O
HN
H
H
H
N
N
O
HO
N
NHBoc
HO
H
O
4
O
N
O
O
O
O
O
d-g
Cl
N
O
O
O
O
H
H
N
5
N
N
N
N
H
N
N
H
S
H
O
O
O
O
5
Reagents and conditions: a) 2,2’-dipyridyldisulfide, DMF, AcOH, 0.2M NaOAc; b) 2-(2-
pyridinyldithio)ethanol p-nitrophenyl carbonate, DMAP, dichloromethane; (c) 2-(2-
pyridinyldithio)ethanol p-nitrophenyl carbonate, DMF, diisopropylethylamine; (d) DCC,
HOSu, DMF, 3-aminopropylmaleimide, diisopropylethylamine; (e) TFA; (f) DM1, DMF, pH
5.6 acetate buffer; (g) 6-maleimidohexanoic acid isobuyl carbonic anhydride, DMF,
pyridine, 0 °C
Synthesis. Initial conjugates were constructed by linking SSTR2 agonist octreotide to
various commercially available chemotoxic payloads. Because of accumulation of the
active agent would be limited by number of copies of SSTR2 expressed at the cell surface,
potent chemotoxic payloads were examined to ensure that payload delivered via
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receptor-mediated endocytosis would be sufficient to kill the tumor cell. Microtubule-
targeting agents DM117, cabazitaxel18, and monomethyl auristatin E19 (MMAE) were
selected as potential payloads; synthesis of the corresponding disulfide-linkable
constructs 1, 2, and 3 are shown in Scheme 1. Because cabazitaxel and MMAE require
linking through non-thiol heteroatoms, a disulfide carbonate or carbamate linker20 was
employed for these payloads. In addition, maleimide 5 containing DM1 linked through a
lyosomally-cleaved Gly-Phe-Leu-Gly sequence21 was synthesized.
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Scheme 2. Synthesis of octreotide, vapreotide, Tyr3-octreotate, and cyclic 6-mer
conjugates
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O
O
STrt
AcO
H
4
5
6
7
8
9
Cl
O
BzO
R1
R1
N
O
OMe
HO
NH2
NH
NH
NH
HO
O
NH
H
O
O
S
O
S
OMe
O
O
H
N
H
N
O
O
O
H
H
O
O
N
O
N
N
S
H
O
S
H
O
a, b
O
O
O
NH
O
O
O
NH
O
R2
R2
O
BocHN
Ph
R3
O
R3
O
S
S
S
S
HN
NH
HN
NH
N
H
N
H
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c, e
O
O
c, d
6a
6a
6a-c
NH
NH
NH
NH
NH2
NHBoc
3
1
2
3
1
2
O
S
octreotide:
6a:
O
S
R =H, R =OH, R =Thr-ol
R =H, R =OH, R =Thr-ol
6b: R1=OH, R2=Me, R3=Trp-NH2
H
N
H
N
1
2
3
vapreotide:
R =OH, R =Me, R =Trp-NH2
H
N
H
1
2
3
Tyr3-octreotate:
R =OH, R =OH, R =Thr-OH
1
2
3
HO
S
H
O
6c:
N
R =OH, R =OH, R =Thr-OH
HO
HO
S
H
O
O
O
O
NH
O
O
O
NH
OH
OH
HO
N
H
O
N
O
H
HN
NH
O
O
HN
NH
H
N
H
N
H
N
N
O
O
O
N
N
O
H
H
O
OH
O
O
O
N
H
7
8
S
NH2
S
NH2
O
O
O
c, f
NH
6a
NH
HN
O
Cl
O
NH
NH
O
N
O
NH
HN
O
HO
O
O
H
N
N
NH
H
O
O
O
O
H
N
O
N
O
O
HO
HO
S
O
O
S
O
O
NH
S
OH
O
N
O
N
O
S
H
HN
NH
O
c, g
N
O
HN
N
H
O
6a
H
O
S
O
O
H
O
NH
NH
S
9
OH
O
c, d
O
S
NH2
OH
H
N
6b-c
N
O
NH
NH
H
Cl
N
HO
HO
S
H
O
2-chlorotrityl resin
O
O
O
O
NH
O
H
N
O
O
OH
Cl
O
O
N
O
H
tBuO
H
HN
NH
N
NHFmoc
h-k
S
H
O
N
N
H
O
S
O
O
NH
O
R1
SH
O
O
S
N
R2
O
S
O
O
10
HN
NH
O
N
H
O
OH
NH
H
NH OH
NH2
O
O
O
NH OH
1
2
11:
R =Me, R =Trp-NH2
1
2
12:
R =OH, R =Thr-OH
O
O
O
NH2
H
N
O
H
N
d
N
N
Me
O
HN
HO
O
H
N
O
H
NH
O
Me
O
HN
HO
NH
O
H
N
NH
O
N
NH
H
H
N
O
H
O
13
14
NH2
NH2
Reagents and conditions: a) BocOSu, DMF, -40 °C to RT; b) S-trityl 3-
mercaptopropionaldehyde, NaBH(OAc)3; c) TFA, triisopropylsilane; d) 1, DMF, pH 7.4
phosphate buffer; e) 2, DMF, pH 5.6 acetate buffer; f) 3, DMF, pH 5.6 acetate buffer; g)
5, DMF, pH 5.6 acetate buffer; h) iterative SPPS with: N-Fmoc-N-Boc-lysine, N-Fmoc-
Nin-Boc-D-tryptophan, Fmoc-tyrosine(tBu), N-Fmoc-N-(S-trityl-3-mercaptopropionate)-
lysine, Fmoc-phenylalanine; i) 1% TFA in DCM; j) HATU, HOAt, DMF, iPr2NEt; k)
95:2.5:2.5 TFA:EDT:water
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Octreotide was then derivatized with a protected thiol for conjugation to the thiol-
reactive payload derivatives (Scheme 2). Selective protection of the lysine side chain with
a Boc group, following by reductive amination with S-trityl-3-mercaptopropionaldehyde
provided 6a. Global deprotection and reaction of the free thiol at the N-terminus with the
appropriate payload provided DM1 conjugate 7, cabazitaxel conjugate 8, MMAE
conjugate 9, and protease-cleavable DM1 conjugate 10. A protected thiol was appended
to the N-terminus of vapreotide22 and Tyr3-octreotate analogously to 7. In addition, cyclic
6-mer peptide 1323 with a pendant thiol was made via solid phase synthesis. Conjugation
of these ligands with DM1 yielded vapreotide conjugate 11, Tyr3-octreotate conjugate 12,
and cyclic 6-mer conjugate 14 respectively.
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Scheme 3. Synthesis of Tyr3-octreotate/DM1 conjugates
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O
2-chlorotrityl resin
HN
OH
O
H
OH
NH
OH
NH
O
O
O
OH
NH
NHBoc
NH2
NH
tBuO
O
TrtS
NHFmoc
O
O
S
NH
H
O
NH2
O
S
H
N
a-e
N
S
O
O
S
N
H
N
H
O
H
N
N
N
HO
HO
O
S
H
O
O
g, h
H
HO
HO
O
S
O
O
O
f
O
S
O
O
N
HO
HO
O
S
H
O
OH
O
OH
O
O
O
O
Cl
N
H
O
OH
N
H
O
N
H
O
HN
NH
HN
NH
N
N
H
HN
NH
H
H
N
O
O
H
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O
17
16
15
NHBoc
OH
NHBoc
OH
O
NH2
OH
Cl
N
O
NHBoc
NH2
NH
NH2
NH
O
STrt
O
NH
H
O
S
N
S
H
N
O
S
O
N
O
H
O
N
H
H
N
g, h
OH
HN
H
O
N
HN
HO
O
S
H
O
O
H
HO
HO
O
S
H
O
O
S
NH
NH
N
O
O
O
NH
HN
HO
O
S
O
O
i, j
OH
O
S
O
O
NH
OH
O
OH
N
H
O
N
H
O
N
O
HN
HN
NH
O
H
N
N
HN
NH
H
H
N
O
O
H
H
O
19
18
Tyr3-octreotate
NHBoc
NH2
NH2
OH
OtBu
OH
NH
NHBoc
NH2
NH
NH2
NH
O
S
NH
H
O
N
H
N
O
O
H
2-chlorotrityl resin
S
H
O
O
N
H
p
NBoc
k-m
O
NH
O
O
N
H2N
O
S
H
O
H
HN
OH
O
OtBu
O
S
O
O
O
N
H2N
O
S
H
O
R
O
OH
O
O
S
O
O
NH
NHFmoc
OH
N
O
HN
NH
H
N
H
H
STrt
O
N
O
S
S
HN
NH
H
O
N
H
O
O
S
S
HN
NH
N
N
O
o
H
N
O
O
20a
20b
(R=OH)
H2N
N
NHBoc
)
n
O
O
22 (PEN-221)
NH2
Cl
21
NH2
(R=
STrt
N
H
O
Reagents and conditions: a) iterative SPPS with: Fmoc-S-trityl-cysteine, Fmoc-
threonine(tBu), N-Fmoc-N-Boc lysine, N-Fmoc-Nin-Boc-D-tryptophan, Fmoc-
tyrosine(tBu), Fmoc-S-trityl-cysteine; b) 95:2.5:2.5 TFA:EDT:water; c) I2, CH3CN/water;
d) Boc2O, iPr2NEt, DMF; e) Et2NH, DCM; f) Boc-O-(S-trityl-2-mercaptoethyl)-D-tyrosine
NHS ester, DMF, iPr2NEt; g) TFA, iPr3SiH; h) 1, DMF, iPr2NEt; i) Boc2O, DMF, iPr2NEt;
j) S-trityl cysteamine HCl, DMF, HATU, iPr2NEt; k) iterative SPPS with: Fmoc-
threonine(tBu), N-Fmoc-N-Boc lysine, N-Fmoc-Nin-Boc-D-tryptophan, Fmoc-
tyrosine(tBu), Fmoc-S-trityl-cysteine, Boc-D-phenylalanine;
l) I2, DMF; m) 20%
trifluoroethanol in DCM; n) HATU, CH2Cl2, S-trityl cysteine amide, iPr2NEt; o) 2,2’-
dithiodipyridine, 1,1,3,3-tetramethyldisiloxane, trifluoroethanol, 12N HCl; p) DM1, THF,
pH 3.7 acetate buffer
Additional Tyr3-octreotate/DM1 conjugates were synthesized by selecting alternative
conjugation points on Tyr3-octreotate that maintained the Tyr-DTrp-Lys-Thr SSTR2
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binding pharmacophore (Scheme 3). Thus, Tyr3-octreotate analogs were synthesized
containing protected thiols on the N-terminal side chain (16), C-terminus (18), or an
activated disulfide bond on the C-terminal side chain (21). Subsequent conjugation of
DM1 provided N-terminal side chain conjugate 17, the C-terminal conjugate 19, and the
C-terminal side chain conjugate 22.
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
Scheme 4. Synthesis of C-terminal side chain conjugates
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20a
OH
NH
OH
e, f
NH2
NH
NH2
OH
NH
c, d
OtBu
a, b
NH2
NH
NHBoc
O
S
O
S
NH
H
H
N
N
O
S
O
S
NH
H
N
H
N
H
g, h
NBoc
N
S
H
O
S
H
O
O
R1
O
O
O
NH
NH
R
O
O
O
R2
R2
H2N
O
S
H
O
OH
OH
H2N
O
S
O
NH
O
O
O
O
O
O
NH
N
H
O
N
H
OH
OtBu
O
S
N
HN
NH
SH
HN
NH
N
H
O
N
H
S
N
N
H
H
O
N
NH
HN
NH
NH2
HN
NH
O
O
N
H
N
H
O
H
O
O
(R =CH2OH, R2 = H)
1
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
24a
24b
24c
26
(R =CONH2, R2 = Me)
1
23a
23b
(R=CO2H)
(R=H)
25
NH2
NH2
O
OH
(R =CONH2, R2 = H)
1
NH2
NHBoc
NH2
NH
OH
O
S
H
N
NH2
O
H
Cl
N
N
H2N
O
S
H
O
O
NH
H
O
O
O
O
O
NH
N
OH
H
l
HN
O
H
O
N
H
O
N
S
O
O
21
R1
O
S
O
O
NH
O
N
S
S
HN
NH
R2
R2
O
N
OH
O
OH
H
O
O
O
N
31
OH
HN
H
O
H
O
O
S
HN
NH
N
O
S
N
H
O
H
O
N
NH2
27-30
O
O
OH
Cl
1
2
i
23a
23b
24a
24b
27
28
29
30
(R =CO2H, R =H)
NH2
24c
NH2
NH
O
1
2
(R =H, R =H)
1
j
2
n
(R =CH2OH, R =H)
O
S
H
N
k
1
2
(R =CONH2, R =Me)
H
O
OMe
OH
NH
MeO
N
H2N
O
O
S
H
O
O
NHBoc
O
O
NH
NH2
OH
O
Ph
O
N
O
H
O
S
NH
O
O
S
HN
NH
H
N
H
S
N
H
OAc
OH
OBz
O
O
H
N
H2N
O
S
H
O
33
O
O
O
OH
m
N
H
O
NH2
26
OH
NH
O
N
NH
HN
NH
N
H
O
NH2
NH
24c
O
o
OH
O
O
O
32
H
N
H
N
O
S
O
N
O
NH2
H
O
N
O
O
N
O
H2N
O
S
O
O
S
O
O
N
HN
OH
H
O O
NH
N
H
O
OH
O
O
N
O
S
S
HN
NH
O
N
H
H
O
O
N
O
34
Cl
NH2
O
Reagents and conditions: a) HATU, CH2Cl2, TrtS-CH2-CH(R)-NH2; b) 2,2’-
dithiodipyridine, 1,1,3,3-tetramethyldisiloxane, trifluoroethanol, 12N HCl; c) HATU,
CH2Cl2, TrtS-CR2 -CH(R1)-NH2 iPr2NEt; d) TFA, thioanisole, water, triisopropylsilane; e)
2
Dap(Dde)-NH2, HATU, CH2Cl2, iPr2NEt; f) hydrazine, DMF; g) maleimidoacetic acid,
HATU, CH2Cl2; h) 92.5:2.5:2.5:2.5 TFA:thioanisole: water:triisopropylsilane; i) DM1, DMF,
pH 4.6 acetate buffer; j) 1, DMF, pH 4.6 acetate buffer; k) 1, DMF, pH 7.4 phosphate
buffer; l) DM4, DMF, pH 7.4 phosphate buffer; m) DM1, DMF, 0.2M NaOAc; n) 2, DMF,
pH 5.6 acetate buffer; o) 3, DMF, pH 5.6 acetate buffer
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9
Additional SSTR2-targeting conjugates linked at the C-terminal side chain were
synthesized as shown in Scheme 4. Modification of the C-terminal amino acid and
appending DM1 resulted in conjugates 27-30. Additional payloads were also appended
onto the C-terminal side chain; conjugation of DM417, cabazitaxel, or MMAE resulted in
conjugates 31, 33, and 34 respectively. In addition, conjugation of DM1 through a
maleimide resulted in conjugate non-cleavable conjugate 32. Finally, a non-binding
control conjugate 37 was synthesized (Scheme 5).
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
Scheme 5. Synthesis of non-binding control
2-chlorotrityl resin
O
O
OH
NH2
NH
NHFmoc
STrt
O
S
H
N
a-c
H
N
H2N
O
S
O
O
O
O
NH
Fmoc
NH
OH
OH
N
H
O
STrt
HN
NH
d-f
N
O
S
NH
H
H
H
O
N
H
36
N
S
H
O
O
O
O
O
NH
HN
HN
O
OH
g, h
HO
HN
NH
N
H
OH
NH
O
NH2
NH
35
O
O
NH2
H
N
NHBoc
H
HN
OH
O
H
N
H2N
O
S
H
O
O
O
S
O
O
OH
O
N
H
O
O
O
S
HN
NH
N
S
N
H
O
O
N
37
O
Cl
H
HN
N
O
NH2
O
Reagents and conditions: a) iterative SPPS with: Fmoc-threonine(tBu), N-Fmoc-N-
Boc lysine, N-Fmoc-Nin-Boc-D-tryptophan, Fmoc-tyrosine(tBu), Fmoc-S-trityl-cysteine,
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Fmoc-D-phenylalanine; b) 92.5:2.5:2.5:2.5 TFA:thioanisole:water: triisopropylsilane; c) I2,
CH3CN/water; d) N-Boc-N’-(4-nitrophenylcarbamate)-1,6-hexanediamine, THF, iPr2NEt,
DMAP, 50 °C; e) S-trityl cysteine amide, DMF, HATU, iPr2NEt; f) piperidine, DMF; g) TFA,
triisopropylsilane; h) 1, DMF, pH 7.4 phosphate buffer
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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
Biological data. Each conjugate was evaluated for SSTR2 affinity by displacement of
[I125]Tyr11-SST14 from isolated membrane fractions of SSTR2-expressing CHO-K1 cells.
In addition, in vitro cytotoxicity of the octreotide conjugates was assessed in SSTR2-
expressing NCI-H524 cells. Each conjugate was incubated with NCI-H524 cells for 2 h
either alone or in the presence of 100 M octreotide, followed by washout and a further
70 h incubation; a typical in vitro experiment is shown in Figure 1. Incubations in the
presence of octreotide were performed to fully occupy cell-surface SSTR2 and prevent
receptor-mediated uptake of the conjugate. A shift in potency under receptor blockade,
compared with conjugate alone, was considered crucial in demonstrating that the
cytotoxic effects of the conjugate are dependent on receptor binding and internalization.
In vitro data for compounds 7-12, 14, 17, 19, and 22 is shown in Table 1.
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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
Figure 1. Payload and conjugate cytotoxicity of 7 and 8 in NCI-H524 cells with and without
receptor blockade, plus their respective payloads
Table 1. SSTR2 binding data and H524 cell assay data for 7-12, 14, 17, 19, 22, payloads,
and SST14
H524
IC50
(nM)b
30
H524 IC50
w/ blockade
SSTR2
Ki (pM)b
Compound
Structurea
(nM)b
7
8
DM1-S(CH2)3-DPhe-[Cys-Phe-DTrp-Lys-Thr-Cys]-Thr-ol
MMAE-CO2(CH2)2SS(CH2)3-DPhe-[Cys-Phe-DTrp-Lys-Thr-Cys]-Thr-ol
cabazitaxel-CO2(CH2)2SS(CH2)3-DPhe-[Cys-Phe-DTrp-Lys-Thr-Cys]-
Thr-ol
120
770
221
150
157
9
260
140
1473
75
560
202
DM1-Mal-GLFG-MC-S(CH2)3-DPhe-[Cys-Phe-DTrp-Lys-Thr-Cys]-Thr-
ol
10
11
12
DM1-S(CH2)3-DPhe-[Cys-Tyr-DTrp-Lys-Val-Cys]-Trp-NH2
DM1-S(CH2)3-DPhe-[Cys-Tyr-DTrp-Lys-Thr-Cys]-Thr-OH
DM1-S(CH2)2CO-[NMeLys-Tyr-DTrp-Lys-Thr-Phe]
DTyr(CH2CH2SDM1)-[Cys-Tyr-DTrp-Lys-Thr-Cys]-Thr-OH
DPhe-[Cys-Tyr-DTrp-Lys-Thr-Cys]-Thr-NHCH2CH2S-DM1
DPhe-[Cys-Tyr-DTrp-Lys-Thr-Cys]-Cys(DM1)-NH2
190
15
22
19
115
757
865
198
528
910
n/a
14
100
15
60
17
15
19
120
22
44
22
10
DM1
MMAE
cabazitaxel
n/a
n/a
n/a
11
3.7
0.26
n/a
n/a
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SST14
5
n/a
n/a
a
Brackets indicate peptide cyclization. Parentheses after amino acids indicate side
b
chain substitution. Mal = 3-aminopropylmaleimide, MC = maleimidocaproyl. All values
determined in duplicate. n/a = not applicable
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
The performance of these conjugates that utilize octreotide as the targeting ligand was
noticeably affected by the payload; while DM1 conjugate 7 showed potency near that of
payload alone, the potency of both cabazitaxel conjugate 8 and MMAE conjugate 9 were
several orders of magnitude worse than their respective payloads. Additionally, only 7
exhibited any signs of receptor-dependent cytotoxicity. The receptor affinity was also
clearly affected by the payload, as both 8 and 9 had lower affinity for SSTR2 than DM1
conjugate 7. While replacement of the disulfide linker with a protease-cleavable linker
(10) did not significantly change the receptor affinity, it was less potent in the NCI-H524
cellular assay, indicating that disulfide linkers might be optimal for release upon SSTR2-
mediated internalization.
While conjugate 7 demonstrated initial signs of receptor-dependent cytotoxicity, the
receptor affinity was significantly worse than native ligand SST14. Given that conjugates
may have to compete against endogenous somatostatin for uptake in SSTR2-expressing
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9
cells, and the knowledge that tumor uptake of small molecule drug conjugates is predicted
to track with receptor affinity8, it was important to examine alternative SSTR2 targeting
ligands in order to improve receptor affinity. The Tyr3-octreotate targeting ligand of
conjugate 12 clearly provided superior receptor affinity compared to octreotide conjugate
7, following the pattern observed with radiolabeled conjugates24. Conjugate 12 also
provided the largest window between cellular potency of the conjugate and potency under
receptor blockade. Vapreotide conjugate 11 appeared nearly as potent in the cellular
assay, but this potency did not shift nearly as much under receptor blockade, indicating
that some of the cellular potency may come from either passive permeability of the more
lipophilic ligand, or conjugate instability and extracellular release of the payload under the
assay conditions. Cyclic 6-mer targeting ligand conjugate 14 did not demonstrate any
noticeable advantages to octreotide conjugate 7 in terms of in vitro properties.
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
Examination Tyr3-octreotate conjugates linked to DM1 at sites other than the N-
terminus show that substitution at either the N-terminal side chain (17) or C-terminal side
chain (22) maintained receptor affinity and cellular potency compared to N-terminal
conjugate 12. Only C-terminal conjugate 19 showed inferior receptor affinity and cellular
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potency among Tyr3-octreotate/DM1 conjugates. In order to differentiate these
conjugates, several compounds evaluated in vivo for their ability to target DM1 to SSTR2-
expressing NCI-H69 tumors. Selected conjugates were administered to NCI-H69 tumor-
bearing mice at 1 mg/kg i.v., while unconjugated DM1 was dosed as a control at 0.4
mg/kg. At 24 h, tumors were excised and treated with excess tris-2-
carboxyethylphosphine (TCEP) to reduce any disulfide bonds. The resulting free thiols
trapped with N-ethyl maleimide (NEM), and total DM1-NEM adduct levels measured.
Values normalized for total DM1 dose are presented in Figure 2. Conjugates 7, 11, and
19, each with SSTR2 affinity >100 pM, exhibited equal, or only slightly superior, DM1
tumor accumulation compared with untargeted DM1. Tyr3-octreotate conjugates 12, 17,
and 22 were superior to other targeting ligand DM1 conjugates. Importantly, conjugate 22
demonstrated the greatest ability to deliver DM1 to an SSTR2-expressing tumor.
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
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7
8
9
0.6
0.4
0.2
0.0
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
7
11
12
14
17
19
22
DM1
Compound
Figure 2. Dose-normalized DM1 levels in NCI-H69 tumors 24 h after conjugate dosing
Table 2. In vitro data for compounds 22, 27-34
OH
NH2
O
S
NH
H
N
S
H
O
R1
O
O
O
NH
NH
R2
R2
OH
N
H
O
R3
HN
NH
N
H
O
NH2
Compoun
d
SSTR2
Ki (pM)b
H524 IC50
(nM)b
H524 IC50 w/
blockade (nM)b
R1
R2
R3
CONH
22
H
S-DM1
22
10
910
2
27
28
CO2H
H
H
S-DM1
S-DM1
13
61
183
10
1597
692
H
CH2O
H
29
30
H
S-DM1
S-DM1
18
15
42
8
1389
1041
CONH
2
M
e
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CONH
31
32
33
34
H
H
H
H
S-DM4
75
27
91
581
2524
14
2
CONH
NH-MalAc-DM1a
140
970
180
2
CONH
SSCH2CH2CO2-
cabazitaxel
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
2
CONH
SSCH2CH2CO2-MMAE
1258
1427
2
a MalAc = maleimidoacetate. b All values determined in duplicate.
Additional conjugates with payloads conjugated at the C-terminal side chain are shown
in Table 2. Modification of the C-terminal amide of 22 to an acid (27) or amino alcohol
(29) maintained receptor affinity, but noticeably reduced potency in NCI-H524 cells.
Complete removal of the C-terminal amide (28) maintained cellular potency, but reduced
receptor affinity.
Slower releasing hindered disulfides were also examined25. Appending a gem-dimethyl
group on the payload side by using DM417 rather than DM1 (31) resulted in reduced
cellular potency and receptor affinity. However, placing the gem-dimethyl group on the
ligand side of the disulfide bond by utilizing penicillamine as the linking amino acid rather
than cysteine (30) showed similar in vitro properties to 22. Non-cleavable DM1 conjugate
32 does demonstrate receptor-dependent cytotoxicity, showing that release of free
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8
payload is not strictly necessary for toxicity, however conjugate 32 was significantly less
potent than disulfide-linked conjugates 22 and 30.
9
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
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51
52
53
54
55
56
57
58
59
60
We investigated if the C-terminal side chain conjugation site of 22 was amenable to
delivery of other microtubule agent payloads. Analogs of 22 where the payload has been
replaced by either cabazitaxel (33) or MMAE (34) shows that the in vitro properties of
conjugates linked at the C-terminal side chain are payload-dependent. The SSTR2
binding affinity of both 33 and 34 were dramatically reduced compared to 22, and neither
exhibited much receptor dependent cytotoxicity. To evaluate if this reduced binding
affinity obtained with other payloads had functional consequences for SSTR2 activity, we
examined 22 and 33 in an SSTR2 internalization assay (DiscoverX) (Figure 3). While
DM1 conjugate 22 is more efficacious than native ligand SST28 at inducing receptor
internalization, the cabazitaxel conjugate 33 was much less potent at inducing receptor
internalization, demonstrating that the inferior receptor affinity of 33 also leads to reduced
SSTR2 agonist activity. Thus, while the C-terminal side chain of Tyr3-octreotate appears
to be an excellent site for DM1 conjugation, the effectiveness of this conjugation site does
not translate for other payloads.
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9
SST28 (EC50 = 2.3 nM)
22 (EC50 = 1.0 nM)
33 (EC50 = 11.0 nM)
1000000
500000
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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0.01
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Concentration (nM)
Figure 3. SSTR2 internalization by 22, 33, and SST28
100
22 (t1/2 = 10.0 min)
30 (t1/2 = 56.8 min)
50
0
0
30 60
120
240
Time (min)
Figure 4. Stability of 22 and 30 in 5 mM GSH at 37 °C
In order to further explore the importance of the release rate of the disulfide linker, 22
and 30 were examined in several additional in vitro and in vivo studies. Stability
experiment of both compounds in 5 mM GSH (Figure 4) demonstrating that 30 possessed
better stability to reducing conditions. However, side-by-side comparisons of both 22 and
30 in beagle dog (Figure 5) shows that the increased stability of the linker does not
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dramatically affect pharmacokinetics. While 30 had a modestly higher AUC (Table 3), this
change is only 1.6-fold, suggesting that the stability of the linker is not the major metabolic
liability of 22. In an SSTR2-expressing NCI-H69 mouse xenograft (Figure 6), both 22 and
30 were able to induce complete regressions at 2.0 mg/kg i.v. without any body weight
loss (see supporting information); however, 22 was more potent at 1.3 mg/kg. Thus, the
improved stability of 30, and its modestly better plasma pharmacokinetics, did not
translate into greater efficacy.
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Figure 5. Beagle dog pharmacokinetics (n=1) of 0.1 mg/kg 22 and 30 i.v.
Table 3. Select beagle dog PK parameters
Parameter
t1/2 (h)
22
30
2.33
1.38
Cmax (μmol/L)
0.708
0.470
0.867
0.769
AUC 0-6h (μmol/L*h)
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Cl (mL/kg/min)
1.66
1.09
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22, 1.3 mg/kg
22, 2.0 mg/kg
30, 1.3 mg/kg
30, 2.0 mg/kg
3000
2000
1000
0
0
10
20
Days
30
40
Figure 6. 22 and 30 in NCI-H69 xenograft efficacy. Arrows indicate dosing days.
In order to demonstrate in vivo receptor dependence of 22, it was compared with
conjugate 37, which is structurally similar but with the lysine residue of the SSTR2 binding
pharmacophore capped with a 1,6-hexanediamine urea. This particular capping group
was selected in order to keep the overall charge and lipophilicity of 37 similar to that of
22. However, this modification of the lysine residue almost completely abolished SSTR2
binding as well as receptor-dependent cytotoxicity (Table 4). Conjugate 37 (0.7 mg/kg
i.v.) was not efficacious in an NCI-H69 xenograft model (Figure 7).
Table 4. SSTR2 binding data and H524 cell assay data of 22 and its non-binding control
37
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Compoun
d
SSTR2
Ki
H524 IC50
(nM)
H524 IC50 w/
blockade (nM)
6
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22
37
22 pM
10
910
644
3.35 M
581
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22, 0.7 mg/kg
37, 0.7 mg/kg
0
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Days
15
20
Figure 7. 22 and 37 in NCI-H69 xenograft efficacy. Arrows indicate dosing days.
Conjugate 22 was tested for binding against all five somatostatin receptor isoforms,
indicating good selectivity for SSTR2 (Table 5). While binding to SSTR2 is close to that
of the native ligand SST14, SSTR5 binding is 30-fold lower than the native ligand, and
binding to all other isoforms is >600-fold lower than the native ligand. In addition, 22 was
examined for selectivity against other GPCRs; 22 was evaluated against a panel of 119
GPCRs, examining for both agonist and antagonist activity. Conjugate 22 was inactive
against all GPCRs in this panel except for SSTR2 and SSTR5 (see supporting
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information). In addition, the passive permeability of 22 was extremely low (Caco-2 A-B
Papp = 0.018x10-6 cm/s), indicating that entry of 22 into non-SSTR2 expressing cells,
either through other receptors or passive diffusion, would be minimal. The activity of 22
was also examined in other SSTR2-positive xenografts. Conjugate 22 demonstrated
complete regressions in HCC-33 small cell lung carcinoma xenografts at 1.0 mg/kg i.v.,
and IMR-32 neuroblastoma xenografts at 1.5 mg/kg i.v. (Figure 8).
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Table 5. SSTR2 selectivity of 22
SSTR1 Kd
310 nM
SSTR2 Kd
16 pM
SSTR3 Kd
19.8 nM
32 pM
SSTR4 Kd
5.48 M
200 pM
SSTR5 Kd
9.6 nM
22
SST14
290 pM
5 pM
320 pM
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Days
Figure 8. 22 in HCC-33 (top) and IMR-32 (bottom) xenografts. Arrows indicate dosing
days.
Conclusion
In summary, we have examined several SSTR2 agonist ligands, conjugation sites, and
microtubule agent payloads for their ability to target payloads to SSTR2-expressing cells.
Optimization for receptor affinity anerd receptor-dependent cellular potency have led to
the discovery PEN-221 (22), a novel maytansinoid-containing conjugated targeted to
SSTR2. Uniquely among SSTR2-targeting peptide conjugates, the payload of 22 is
conjugated at the C-terminal side chain, which allows for good receptor affinity and
excellent levels of DM1 delivered into SSTR2-expressing tumors. Conjugate 22 has
excellent affinity for SSTR2, is very potent at inducing receptor internalization, and has
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good selectivity for SSTR2 over other somatostatin receptor subtypes as well as other
GPCRs. PEN-221 (22) is able to induce regressions in several SSTR2-expressing
xenograft models, and is currently being evaluated in human clinical trials.
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Experimental section
Chemistry general methods. DM1, DM4, and MMAE were purchased from Ontario
Chemicals, cabazitaxel was purchased from Indena, octreotide and vapreotide were
purchased from ChemPep, Tyr3-octreotate was synthesized at ChemPartner, and all
other reagents were purchased from Chem-Impex, Aldrich, Fisher, or Ark Pharm, and
used without further purification. Solid phase peptide synthesis was carried out on a CEM
Liberty Blue peptide synthesizer. All silica gel chromatography was carried out on
RediSep Rf Gold silica gel disposable flask columns (20-40 micron) on a Teledyne Isco
combiflash system. Reverse phase chromatography was carried out on RediSep Rf Gold
C18 reverse phase columns (20-40 micron), and preparative HPLC purification was done
on a Waters Sunfire C18 OBD Prep HPLC column, 100 Å, 5 m, 30 x 150 mm. Final
compounds were lyophilized on an SP Scientific Benchtop Pro 9 EL lyophilizer. Chemical
purities were >95% for all final compounds, as assessed by an Agilent 1100 LCMS with
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an Agilent Eclipse Plus C18 column, 3.5 m, 4.6 x 100 mM, with detection at 254 and 280
nm. Mass spectrometry data were gathered on an Agilent G1946D mass spectrometer
with electrospray ionization. Procedures for synthesis of compounds 1-19, and 23-37 are
in the supporting information.
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Radioligand binding assays. Radioligand binding assays were performed at Eurofins
Discovery Services, Taipei. CHO-K1 cells stably transfected with a plasmid encoding the
desired human somatostatin receptor were used to prepare membranes in modified
HEPES pH 7.4 buffer using standard techniques. An aliquot (1 µg for SSTR1, 0.1 µg for
SSTR2, 0.375 µg for SSTR3, 0.25 µg for SSTR4, 2 µg for SSTR5) of membrane was
incubated with 0.03 nM (SSTR2) or 0.1 nM (all other SSTRs) [125I]somatostatin for either
240 minutes (SSTR2) or 120 minutes (all other SSTRs) at 25 °C. Non-specific binding
was estimated in the presence of 1 µM somatostatin. Membranes were filtered and
washed 3 times and the filters counted to determine [125I]somatostatin specifically bound.
Compound treatment was done at six concentrations, with 10 nM as highest
concentration and five-fold dilutions for SSTR2, 10 M and 3-fold dilutions for SSTR1 and
SSTR4, or 1 M and 10-fold dilutions for SSTR3 and SSTR5.
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Cytotoxicity assay. NCI-H524 (ATCC) human lung cancer cells were plated in 96 well,
V-bottomed plates (Costar) at a concentration of 5,000 cells/well and 24 hours later were
treated with compound for 2 hours. Compound starting dose was 10 M and three-fold
serial dilutions were done for a total of ten points. For experiments with octreotide
competition, cells were first treated with 100 M octreotide acetate (Chempep) for 30 min,
then treated with compound as above. After 2 hr compound treatment, cells were spun
down, the drug containing media was removed and fresh complete medium was added
and used to resuspend the cells, which were spun again. After removal of the wash
media, the cells were resuspended in complete medium, then transferred into white
walled, flat bottomed 96 well plates. Cells were further incubated for an additional 70
hours to measure inhibition of cell proliferation. Proliferation was measured using Cell
Titer Glo reagent using the standard protocol (Promega) and a Glomax multi + detection
system (Promega). Percent proliferation inhibition was calculated using the following
formula: % inhibition = (control-treatment)/control*100. Control is defined as vehicle
alone. IC50 curves were generated using the nonlinear regression analysis (four
parameter) with GraphPad Prism 6.
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