Enhancing the Pharmacokinetics and Antitumor Activity of an α-Amanitin-Based Small-Molecule Drug Conjugate via Conjugation with an Fc Domain

Francesca Gallo, Barbara Korsak, Christoph Mu

Article

Enhancing the Pharmacokinetics and Antitumor Activity of an

α‑Amanitin-Based Small-Molecule Drug Conjugate via Conjugation

with an Fc Domain

Olga Avrutina, Harald Kolmar, and Andreas Pahl*

ller, Torsten Hechler, Desislava Yanakieva,

Cite This: J. Med. Chem. 2021, 64, 4117 −4129

Read Online

ACCESS

Metrics & More

Article Recommendations

sı Supporting Information

ABSTRACT: Herein we describe the design and biological

evaluation of a novel antitumor therapeutic platform that combines

the most favorable properties of small-molecule drug conjugates

(SMDCs) and antibody drug conjugates (ADCs). Although the

small size of SMDCs, compared to ADCs, is an appealing feature

for their application in the treatment of solid tumors, SMDCs

usually suﬀer from poor pharmacokinetics, which severely limits

their therapeutic eﬃcacy. To overcome this limitation, in this

proof-of-concept study we grafted an α-amanitin-based SMDC that

targets prostate cancer cells onto an immunoglobulin Fc domain

via a two-step “program and arm” chemoenzymatic strategy. We

demonstrated the superior pharmacokinetic properties and

therapeutic eﬃcacy of the resulting Fc-SMDC over the SMDC in a prostate cancer xenograft mouse model. This approach may

provide a general strategy toward eﬀective antitumor therapeutics combining small size with pharmacokinetic properties close to

those of an ADC.

INTRODUCTION

required to turn SMDCs into eﬀective anticancer therapeutics.

Conjugation to Fc fragments has been successfully applied to

limit renal clearance of small molecules by increasing their

molecular size and exploiting the Fc receptor (FcRn) recycling

process responsible for the long half-life of immunoglobulin G

■

Antibody drug conjugates (ADCs), which use monoclonal

antibodies as tumor-homing vehicles for site-speciﬁc delivery

of cytotoxic drugs to the tumor tissue, represent a break-

through in cancer treatment. By recognizing tumor-associated

,10

antigens with high speciﬁcity, ADCs deliver cytotoxic drugs to

the tumor site and reduce the systemic toxicities often caused

by conventional chemotherapy. Despite the success of

trastuzumab-emtansine in breast cancer treatment, the clinical

implementation of ADCs for the therapy of solid malignancies

is still hampered by the poor penetration of full format

antibodies (IgGs) in circulation.

Introduction of an Fc

portion in the structure of active pharmacological proteins or

peptides such as etanercept and romiplostin enabled their

therapeutic use by optimization of pharmacokinetic properties,

namely, prolongation of circulatory half-life. In this proof-of-

concept study, we grafted a SMDC onto an IgG1-Fc scaﬀold

and compared the therapeutic eﬃcacy of this novel small-

molecule-Fc-drug conjugate (Fc-SMDC) with that of a PSMA-

targeting SMDC lacking the Fc portion. We hypothesized that

by grafting the SMDC onto an IgG1-Fc scaﬀold, the novel

product would display a prolonged half-life in circulation

compared to the SMDC, allowing for a gradual cytotoxic drug

antibodies in the solid tumor tissue. Small-molecule drug

conjugates (SMDCs) based on small organic ligands as tumor-

homing vehicles have been recently gaining growing attention

due to their advanced tumor penetration, in contrast to high-

molecular-weight ADCs. Promising results have been achieved

in the preclinical setting with SMDCs targeting a limited

number of receptors, i.e., the folate receptor, the prostate-

speciﬁc membrane antigen (PSMA),

the somatostatin

receptors, and the carbonic anhydrase IX (CIX).

Received: January 1, 2021

Published: March 23, 2021

On the one hand, SMDCs might penetrate solid tumor

tissue better due to their small molecular size compared to

ADCs. On the other hand, the small size of SMDCs is the main

reason for their extremely short half-life in circulation, which in

turn limits their gradual tumor uptake and compromises their

therapeutic eﬃcacy. Prolongation of the circulatory half-life is

2021 American Chemical Society

117

J. Med. Chem. 2021, 64, 4117−4129

Article

Figure 1. Structures of the SMDC and Fc-SMDC targeting PSMA. Molecules are color-coded as follows: PSMA-targeting ligand, L-Glu-urea-L-Glu

DUPA, blue); PSMA supporting−binding spacer 8-Aoc-Phe-Phe (Pep, blue); thiosuccinimide (HDP 30.2284, cyan) or triazole (HDP 30.2972,

magenta) linkage; cathepsin B cleavable linker Val-Ala (green); PAB self-immolative spacer (orange); and α-amanitin (red).

(

accumulation in the tumor tissue and enhanced antitumor

eﬃcacy.

amanitin. α-Amanitin is a bicyclic octapeptide isolated from

the green death cap mushroomAmanita phalloides, which acts

To the best of our knowledge, this is the ﬁrst report

proposing the chemical grafting onto the Fc scaﬀold of both: a

targeting moiety for providing target selectivity and a cytotoxic

payload for exerting an antitumor eﬀect. The constructs

investigated in the present study have been designed to target

prostate cancer (PCa) cells via a glutamate-urea-based

targeting motif that binds to the prostate-speciﬁc membrane

antigen (PSMA) with high aﬃnity. PSMA is a transmembrane

as a potent RNA polymerase II inhibitor in eukaryotic cells,

signiﬁcantly slowing down the rate of transcription. In

contrast to the majority of applied payloads, α-amanitin kills

both dividing and nondividing cells enabling the respective

conjugates to eradicate dormant tumor cells and thus

preventing tumor recurrence and metastasis. Additionally, its

hydrophilic nature excludes cell permeation via passive

diﬀusion and requires receptor-mediated internalization to

glycoprotein overexpressed by virtually all PCas and on the

exert its cytotoxic activity. This makes α-amanitin appealing

neovasculature of other solid tumors. The same PSMA-

from the safety perspective.

We expected that the

targeting motif was explored in clinical trials with a tubulysin

combination within a single platform of (1) a small organic

ligand with eﬃcient targeting and internalization properties,

B-based SMDC product, EC1169. PSMA-targeted ADCs

based on microtubule inhibitors and DNA-cross-linkers were

also investigated in the clinical setting of prostate cancer

(

2) a hydrophilic payload possessing a unique mode of action,

and (3) an Fc portion conferring extended circulatory half-life

would lead to an ADC-like therapeutic agent with eﬀective

antitumor activity yet much smaller in size.

15,16

therapy with limited success.

The discontinuation of clinical development of some ADCs

based on microtubule inhibitors for solid cancer indications

prompted us to investigate a toxic payload with an alternative

RESULTS

■

mode of action distinct from currently used toxic payloads

and more favorable physicochemical properties, namely, α-

The design of an SMDC construct targeting PSMA started

with the selection of a small molecule capable of binding

118

J. Med. Chem. 2021, 64, 4117−4129

Journal of Medicinal Chemistry

Article

Scheme 1. Synthesis of the PSMA-Targeting Sequence

Reagents and conditions: (a) H-Glu(O Bu)-O Bu, DSC, TEA, DMF, and 0 °C; (b) H-Glu(OBn)-O Bu, TEA, DMF, 0 °C to rt, and 77%; (c) H ,

Pd-C, EtOAc, rt, and 96%; (d) 7, HBTU/HOBt, DIPEA, 40 W, and 60 °C; and (e) TFA/TIPS/H O/DTT, rt, and 68%.

Scheme 2. Synthesis of SMDC 1 and the Drug-Linker Payload 12

Reagents and conditions: (a) 10, Cs CO₃_a_q, DMA, Ar, rt, and 62%; (b) (1) TFA, 2 min, and rt; (2) NH₃_a_qpH 10, and rt; and (3) DBCO-NHS,

DIPEA, DMF, Ar, rt, and 86%; (c) (1) TFA, 2 min, and rt; (2) NH₃_a_qpH 10; and (3) ECMS, DIPEA, DMF, Ar, rt, and 86%; and (d) 8, DIPEA,

DMSO, Ar, rt, and 43%.

PSMA and enhancing its constitutive internalization. The

glutamate-urea-based ligand DUPA (2-[3-(1,3-dicarboxyprop-

yl)-ureido]pentanedioic acid) was proven to bind to PSMA

with high aﬃnity (K = 8 nM; IC = 47 nM) and carry its

i 50

cargo into PSMA-expressing cells via receptor-mediated

endocytosis.

119

J. Med. Chem. 2021, 64, 4117−4129

Article

Figure 2. Strategy for programming and arming the Fc scaﬀold and characterization of Fc-SMDC HDP 30.2972. (A) Step a: the IgG1-Fc-LPETGG

scaﬀold (14) is programmed by attaching at the C-terminus the trifunctional linker 15 (B) containing the DUPA-Pep PSMA binding sequence

(

blue) and an azide as “clickable” handle (magenta) through sortase A (eSrtA)-mediated ligation; step b: the programmed DUPA-Pep-Fc 16 is

armed with the dibenzocyclooctine (DBCO)-Val-Ala-PAB-α-amanitin payload (12) via strain-promoted azide-alkyne cycloaddition (SPAAC).

HRESI-MS analysis after deconvolution of (C) IgG1-Fc-LPETGG (14), (D) DUPA-Pep-Fc 16, and (E) DUPA-Pep-Fc-α-amanitin Fc-SMDC (2,

HDP 30.2972). (F) SDS-PAGE analysis under reducing and nonreducing conditions and (G) anti-α-amanitin Western blot under nonreducing

conditions. SDS-PAGE was performed on Fc-LPETGG (14; lane 1), DUPA-Pep-Fc (16; lane 2), and DUPA-Pep-Fc-α-amanitin Fc-SMDC (2,

HDP 30.2972, lane 3), followed by staining with Coomassie blue or Western blot analysis with immunodetection of α-amanitin. Pep = 8-Aoc-Phe-

Phe, LPR = linker-to-protein ratio, and DPR = drug-to-protein ratio.

A growing body of evidence shows that the overall binding

to PSMA can be enhanced by introducing a supporting−

binding spacer that ﬁts the structural contour and chemical

environment of the gradually narrowing 20 Å tunnel, which

accesses the PSMA binding site. The supporting−binding

spacer 8-Aoc-Phe-Phe, henceforth referred to as Pep, as shown

by Kularatne et al., enhances the binding aﬃnity of a DUPA

analogue by a factor of 6 and therefore was used in

combination with DUPA (DUPA-Pep) for targeting

30.2972 (2), we used the more stable triazole linkage instead

of the thiosuccinimide (Figure 1, shown in magenta) to anchor

the α-amanitin payload with the Fc framework. Furthermore,

to avoid steric interference with the large Fc protein, a ﬂexible

EG (ethylene glycol) dimer was introduced as spacer between

the targeting motif and the drug-linker payload. The

hydrophilicity of the (EG ) spacer provided an additional

advantage by increasing the water solubility of the synthetic

component enabling conjugation in aqueous media. As

outlined in Scheme 1, the DUPA precursor (6) was

synthesized according to the procedure reported by Kularatne

PSMA. As shown in Figure 1, the SMDC HDP 30.2284

1) comprises the binding sequence DUPA-Pep (shown in

(

blue) conjugated to the cytotoxic drug payload (shown in red)

via a thiosuccinimide linkage (shown in cyan) through a

dipeptide Val-Ala linker (shown in green). The dipeptide Val-

Ala is a substrate for the lysosomal cathepsin B, a proteolytic

et al. with minor modiﬁcations toward easier handling

conditions. The commercially available tert-butyl glutamic acid

3 was ﬁrst activated as succidimidyl ester 4. Following the

addition of γ-benzylated glutamic acid, the fully protected

DUPA intermediate 5 was hydrogenated yielding the DUPA

precursor 6. Acylation of the resin-bound peptide 7

(synthesized using Fmoc-based solid-phase peptide chemistry)

with precursor 6 was followed by resin cleavage and global

deprotection yielding the DUPA-Pep binding sequence 8. The

binding sequence 8 bears a C-terminal cysteine to address the

maleimide-containing drug-linker payload. The approach to

access SMDC 1 is outlined in Scheme 2. The key step is the

formation of an ether bond through alkylation of the phenolic

20,21

enzyme overexpressed in tumor cells.

To ensure cleavage

of the Val-Ala motif for release of the free toxin, a self-

immolative p-aminobenzyloxy spacer (PAB; Figure 1, shown in

orange) was introduced between the toxin and the dipeptide

linker. The SMDC grafted onto the IgG1-Fc scaﬀold was

designed as an HDP 30.2284 analogue. However, thiosucci-

nimide-linked conjugates might be susceptible to drug loss

over time through a retro-Michael addition or thiol exchange

reactions in thiol-containing environments. Thus, in HDP

120

J. Med. Chem. 2021, 64, 4117−4129

Journal of Medicinal Chemistry

′-hydroxy group in the tryptophan residue of natural α-

Article

strated high in vitro activity in PSMA-positive cells. HDP

30.2284 (1) displayed 555-fold higher activity than the

unconjugated α-amanitin (IC₅₀0.863 vs 479 nM) (Figure

3 A,C). Compared to HDP 30.2284 (1), Fc-SMDC HDP

amanitin with bromide 10. Following alkylation, the α-

amanitin derivative 11 was subjected to a two-step protocol

involving global deprotection and functionalization with N-

maleimidocaproyl-oxysuccinimide ester (ECMS). The result-

ing α-amanitin derivative 13 was then coupled with the DUPA-

Pep sequence 8 aﬀording SMDC HDP 30.2284 (1) in 43%

yield. For grafting the SMDC product onto a human IgG1-Fc

scaﬀold, a two-step “program and arm” strategy (Figure 2) was

developed.

The IgG1-Fc scaﬀold was ﬁrst programmed by attaching the

trifunctional linker 15 simultaneously bearing the cell-speciﬁc

targeting motif DUPA-Pep (shown in blue), an N-terminal

oligoglycine substrate for the regiospeciﬁc labeling of the Fc

scaﬀold (purple), and a clickable handle (magenta, Figure 2B)

for subsequent arming with the toxin payload 12. To generate

a product with a deﬁned drug-to-protein ratio (DPR) of 2, the

human IgG1-Fc scaﬀold was equipped at the C-termini with

the peptide tag LPETGG allowing for Fc programming via

sortase A (SrtA)-mediated ligation. SrtA is a transpeptidase

from Staphylococcus aureus widely used for site-speciﬁc

24−27

modiﬁcations of antibodies and antibody fragments

that

catalyzes the formation of a new amide bond between the C-

terminal sorting motif LPXTG (where X is equal to any amino

acid) and an N-terminal oligoglycine (G)n (n = 3−5)

nucleophile. The programmed DUPA-Pep-Fc conjugate (16,

Figure 2A) was conﬁrmed to be a disulﬁde-linked Fc dimer by

SDS-PAGE under reducing and nonreducing conditions

(Figure 2F). HRESI-MS analysis under nonreducing con-

ditions (Figure 2D) further conﬁrmed the expected molecular

weight for an Fc dimer. Deconvolution results revealed two

diﬀerent peaks, which were assigned to versions of the Fc

dimer modiﬁed with one to two molecules of the trifunctional

linker 15, resulting in an average linker-to-protein ratio (LPR)

of 1.62 (Figure 2D). Following the SrtA-mediated ligation, the

strain-promoted azide-alkyne cycloaddition (SPAAC; Figure

Figure 3. Cytotoxicity of SMDC HDP 30.2284, Fc-SMDC HDP

0.2972, and unconjugated α-amanitin in the PSMA-positive LNCaP

(PSMA +++) cell line. Both conjugates were tested in the presence or

the absence of 2-PMPA excess, a competitive inhibitor of PSMA. (A)

Dose-response cytotoxic potential of HDP 30.2284. (B) Dose-

response cytotoxic potential of HDP 30.2972. (C) Tabular summary

of the IC₅₀values (data shown are mean ± SEM; n = 3).

A, step b) was explored to conjugate the drug-linker

component 12 (Scheme 2). The chemoselective copper-free

click chemistry was chosen to minimize protein oxidation by

reactive oxygen species and to avoid residual copper in the ﬁnal

product, therefore preventing potential copper-related cytotox-

icity. As shown in Scheme 2, the drug-linker component 12

was obtained in 86% yield from the intermediate 11 upon

global deprotection and coupling to DBCO-NHS ester.

Incorporation of α-amanitin in the ﬁnal product 2 (HDP

30.2972 (2) was approximately 17-fold less active, showing an

IC of 15.2 nM (Figure 3). To further conﬁrm that activity of

both PSMA-targeted conjugates (1 and 2) was receptor-

mediated, the cytotoxic potential of the conjugates was

determined in the presence of a 100-fold (HDP 30.2284, 1)

or a 200-fold (HDP 30.2972, 2) molar excess of 2-PMPA, a

well-known inhibitor of PSMA enzymatic activity and a

30.2972) was conﬁrmed by SDS-PAGE under nonreducing

conditions (Figure 2F), showing signal migration to higher

molecular weight (lane 3) in comparison to DUPA-Pep-Fc

competitive inhibitor of DUPA. Under these conditions, the

(16; lane 2) and by Western blot analysis with immunode-

cytotoxic activity of both conjugates in LNCaP cells was

completely inhibited. Further biological characterization of

conjugates HDP 30.2284 (1) and HDP 30.2972 (2) was

focused on in vivo studies: determination of the maximum

tolerated dose, pharmacokinetics and biodistribution, and

antitumor eﬃcacy. In mice, the maximum tolerated dose

(MTD) was found to be 0.046 mg/kg (18.74 μg/kg of α-

amanitin) for HDP 30.2284 (1) and 1.0 mg/kg (25.7 μg/kg of

α amanitin) for HDP 30.2972 (2). Biodistribution of both

conjugates was monitored to follow blood pharmacokinetics

and accumulation in tumor, liver, and kidneys. For

pharmacokinetic analyses, animals were injected with a dose

corresponding to 4× MTD (0.184 mg/kg) of HDP 30.2284

(1) to secure detectable concentrations of the conjugate in the

serum and the organ extracts. For the quantitation of the

tection of α-amanitin (Figure 2G). Heterogeneity of the

DUPA-Pep-Fc precursor 16 with respect to the number of

attached linker molecules led to the formation of heteroge-

neous species with a drug-to-protein ratio (DPR) ranging from

(

to 2, as conﬁrmed by the deconvoluted mass spectrum

Figure 2E). The average DPR was calculated to be 1.72,

consistent with the LPR value reported for precursor 16

Figure 2D). Importantly, no species loaded with the linker but

not conjugated to the toxin, which could compete with HDP

0.2972 for binding to either FcRn or PSMA, were detected.

(

Initial in vitro characterization of SMDC HDP 30.2284 (1)

and the chemically programmed and armed Fc-SMDC HDP

0.2792 (2) was carried out in the LNCaP cell line

overexpressing the targeted receptor. Both conjugates demon-

121

J. Med. Chem. 2021, 64, 4117−4129

Article

Figure 4. Pharmacokinetic proﬁle and biodistribution of SMDC HDP 30.2284 and Fc-SMDC HDP 30.2972. (A) Blood pharmacokinetics of HDP

0.2284 with the indicated t₁_/₂after single-dose administration of 0.184 mg/kg (CB17-Scid mice, mean concentration ± SEM, n = 3). (B)

Biodistribution of HDP 30.2284 in the tumor, liver, and kidney after single-dose administration of 0.184 mg/kg (CB17-Scid xenografted with

LNCaP tumors, mean concentration ± SEM, n = 3). (C) Blood pharmacokinetics of HDP 30.2972 after single-dose administration of 1 mg/kg,

(

CB17-Scid xenografted with LNCaP tumors, mean concentration ± SEM, n = 3). (D) Biodistribution of HDP 30.2972 after single-dose

administration of 1 mg/kg (CB17-Scid xenografted with LNCaP tumors, mean concentration ± SEM, n = 3). (E) Concentration of free α-amanitin

released from HDP 30.2972 after a single i.v. dose of 1 mg/kg in the tumor, liver, and kidney (CB17-Scid xenografted with LNCaP tumors, mean

concentration ± SEM, n = 3). No free α-amanitin was detected in the serum after administration of HDP 30.2972 (data not shown).

conjugate, we used an anti-α-amanitin ELISA, which detects

the parent compound HDP 30.2284, as well as all possible

metabolites presenting α-amanitin in the structure. Serum

levels of HDP 30.2284 (1) rapidly declined and were below

the lower limit of quantiﬁcation 24 h and 48 h after

administration (Figure 4A,B). The concentration of HDP

μg/g was observed 4 h after single-dose administration and was

followed by a slight drop to approx. 1.0 μg/g, detectable 48 h

after administration. These results suggested a high accumu-

lation of the conjugate in the kidney. The in vivo

pharmacokinetics of Fc-SMDC HDP 30.2972 (2) was

determined at a dose corresponding to the MTD (1 mg/kg).

The pharmacokinetic analysis revealed that with a size lower

than that of a conventional ADC (approx. 61 vs 150 kDa,

respectively), Fc-SMDC HDP 30.2972 (2) showed ADC-like

pharmacokinetic properties and a much longer circulatory half-

life compared to SMDC HDP 30.2284 (1). Fc-SMDC HDP

30.2972 (2) displayed a biphasic elimination proﬁle typical for

molecules harboring an Fc motif (Figure 4C). Fast (α-phase)

and slow (β-phase) elimination half-lives were determined to

be 26 min and 172.6 h (approx. 7.2 days), respectively (Figure

4C).

30.2284 (1) in the tumor reached its maximum (approx. 50

ng/g) 1 h after administration, and a much lower

concentration of only approx. 2 ng/g 48 h after administration

(Figure 4B). These results suggested that in a subsequent in

vivo antitumor eﬃcacy study, a high administration frequency

should be applied to increase the exposure and provide higher

levels of tumor accumulation. Overall, a high conjugate

concentration was found in the liver reﬂecting the blood

concentration due to the high congestion of this organ. Despite

complete elimination from circulation, a concentration of

approx. 20 ng/g was detected in the liver 48 h after

administration. In contrast to the low concentrations of the

toxin detected in the tumor and the liver, very high

concentrations were observed in the kidney throughout

duration of the study. A peak concentration of approx. 2.0

In the liver and the kidney, a low concentration throughout

duration of the study was detected with a transient increase

observed at day 7 (Figure 4D). Detectable levels of intact HDP

30.2972 (2) were present in the tumor only 5 and 15 min after

administration. For the remaining time-points, the concen-

122

J. Med. Chem. 2021, 64, 4117−4129

Article

Figure 5. In vivo antitumor activity and tolerability of SMDC HDP 30.2884 and Fc-SMDC HDP 30.2972. (A) Eﬃcacy of the therapy with HDP

0.2284. (B) Tolerability of HDP 30.2284. (C) Eﬃcacy of the therapy with HDP 30.2972. (D) Tolerability of HDP 30.2972. (E) Long-term

survival follow-up of the animals treated with HDP 30.2972. The mean tumor volume ± SEM is shown for the eﬃcacy experiment and the median

body weight ± SEM for tolerability graphs (n = 8−9).

tration of intact conjugate in the tumor was below lower limit

of quantiﬁcation. The Val-Ala-PAB linker was designed to be

cleaved inside of the cells via intracellular cathepsin B. To

determine the concentration of the released payload, the

concentration of free α-amanitin in the same tissue samples

was measured using an anti-α-amanitin ELISA. The serum

concentration of the released toxin was below lower limit of

quantiﬁcation throughout the study duration, which is

evidence of high conjugate stability in the circulation (data

not shown). Only minimal variation in α-amanitin levels over

time was observed in the liver and the kidney (Figure 4E). The

maximum concentration of 14 ng/g was measured in the

kidney 72 h after HDP 30.2972 (2) administration and

remained at a similar level up to 14 days following

administration. The concentration of the unconjugated toxin

detected in the kidney was slightly higher than that in the liver

the released toxin was found in the tumor with the peak

concentration of approx. 54 ng/g measured at days 1 and 3

after administration. At day 7, the tumor toxin concentration

dropped slightly to 33 ng/g but was still detectable in the

tumor tissue even 14 days after single-dose administration

(

Figure 4E). Next, the therapeutic activities of both SMDC (1)

and Fc-SMDC (2) were tested in CB17-Scid male mice

implanted with PSMA-positive LNCaP tumors. Frequent

dosing of HDP 30.2284 (1) was applied to provide high

exposure and compensate for the short half-life of this

compound (Figure 5A). Treatment with HDP 30.2284 (1)

led to limited tumor regression at all doses, with the lowest

eﬃcacy observed at 0.023 mg/kg (1/2 MTD) 2×/week, while

the intermediate eﬃcacy and the highest antitumor activity

were observed at 0.0115 mg/kg (1/4 MTD) 5×/week and

0.0115 mg/kg (1/4 MTD) 3×/week, respectively (Figure 5A).

(Figure 4E). Most importantly, the highest concentration of

123

J. Med. Chem. 2021, 64, 4117−4129

Journal of Medicinal Chemistry

Article

All dosing regimens were well tolerated as indicated by only

minor losses in the relative body weight (Figure 5B). Despite

transient tumor-growth inhibition, after cessation of the

therapy, tumors in all groups treated with HDP 30.2284 (1)

started to regrow with kinetics similar to that of the vehicle-

injected group. Doses and the frequency of HDP 30.2972 (2)

administration were based on the pharmacokinetic proﬁle and

MTD studies of this compound (Figure 5C,5D). The

antitumor eﬀect of HDP 30.2972 (2) was dose-dependent

This is in contrast to the majority of currently used in the

development of ADCs and SMDCs payloads, which are highly

hydrophobic and able to induce cytotoxicity and antitumor

activity even in the absence of receptor-mediated internal-

ization as a consequence of possible premature linker cleavage

29,30

and passive uptake.

Premature linker cleavage and release

of the hydrophobic cytotoxic payload is the main reason for

oﬀ-target toxicities. In the case of ADCs, the conjugation of

such payloads to large hydrophilic antibodies via stable linkers

can mitigate unspeciﬁc uptake and reduce the risk of systemic

toxicity. On the contrary, the conjugation to low-molecular-

weight ligands is not expected to have an inﬂuence on the

payload physicochemical properties suﬃcient to avoid an

unspeciﬁc uptake. In several studies, it was demonstrated that

SMDCs featuring hydrophobic toxins such as tubulysin B,

MMAE, or paclitaxel show cytotoxic potentials in a

concentration range similar to those observed for the

(

Figure 5C).

While a dose of 1 mg/kg led to complete remission, the 0.25

mg/kg dose was hardly eﬀective, and the 0.5 mg/kg dose

showed intermediate eﬃcacy (Figure 5C). Furthermore, a

dosing frequency-dependent eﬀect was observed in the groups

dosed with 0.25 mg/kg, whereas the three-times-per-week

regimen led to the highest tumor response. The tumor volume

of the group administered with 0.5 mg/kg once per week

converges with the tumor volume of the group administered

with 0.25 mg/kg three times per week, but 0.25 mg/kg twice

per week was less eﬃcacious than 0.5 mg/kg once a week

,31,32

unconjugated toxins,

supporting the hypothesis of passive

toxin cellular uptake despite conjugation to a small targeting

molecule. However, in our experiments, a remarkably lower

potential was observed in PSMA-negative PC3 cells for both

tested compounds, further conﬁrming a PSMA-mediated

uptake (Supporting Information Figure S4). Cytotoxicity

experiments performed in the presence of the competitive

inhibitor 2-PMPA ultimately conﬁrmed that receptor-mediated

internalization is essential to unfold the cytotoxic potential of

α-amanitin. To the best of our knowledge, α-amanitin-based

SMDCs described in the literature so far demonstrated only

4−5-fold better targeting properties compared to the

(

Figure 5C). Although a very similar tumor response was

observed for 1 mg/kg administered once per week and 0.5 mg/

kg administered twice per week, a higher number of durable

complete responses was observed for the animals dosed with 1

mg/kg. In the long-term follow-up, all animals dosed with 0.5

mg/kg twice per week experienced tumor relapse and had to

be sacriﬁced due to unacceptably high tumor volume (Figure

E). In contrast, at day 116 following initiation of the therapy,

out of 8 animals from the group treated once per week with 1

33,34

mg/kg were still alive, demonstrating complete response and

unconjugated toxin.

Here, we report for the ﬁrst time a

signiﬁcantly longer median survival (Figure 5E).

tremendous increase in the activity of α-amanitin upon

conjugation to a small-molecule tumor-targeting ligand. A

comparative pharmacokinetic and biodistribution study

revealed signiﬁcant diﬀerences in the half-life, tumor, and

organ accumulation of SMDC (1) and Fc-SMDC (2)

products. The small size of HDP 30.2284 (1) and the

consequent rapid glomerular ﬁltration accounts for its short in

vivo half-life of only approx. 44 min (Figure 4A). For Fc-

SMDC (2), a blood pharmacokinetic proﬁle typical for

molecules presenting an Fc motif in the structure was observed

(t1/2 α-phase = 26 min and t1/2 β-phase = 7.2 days). As the

molecular weight of HDP 30.2972 (2) slightly exceeds the

renal cutoﬀ of 60 kDa, the contribution of renal ﬁltration to its

clearance is supposed to be lower than for HDP 30.2284.

However, engagement of Fc-SMDC (2) with the FcRn

recycling process, which protects the molecule from proteolytic

degradation, retains it in the intracellular compartment and

concomitantly protects it from rapid renal clearance and is thus

supposed to be most likely the main contribution to the

DISCUSSION

■

Despite representing a breakthrough in cancer treatment,

challenges still remain for application of ADCs to diﬃcult-to-

treat solid tumors, due to the speciﬁc tumor physiology and

poor ADC penetration in the tumor mass. From this

perspective, SMDCs may potentially achieve better penetration

in the tumor tissue. Additionally, the design of a conjugate that

is able to eradicate the tumor and overcome tumor-resistance

and oﬀ-target toxicity issues may beneﬁt from the application

of payloads with alternative modes of action and more

favorable physicochemical properties.

In the present study, we combined the tumor-homing

properties of the small-molecule DUPA targeting PSMA with

the novel mode of action of α-amanitin, a potent RNA

polymerase II inhibitor, for the treatment of PCa. However,

SMDCs suﬀer from a short circulation half-life because of their

small size, limiting the conjugate tumor uptake and eﬃcacy.

Hence, an attempt was made to design a small format

conjugate with improved pharmacokinetic properties and

therapeutic activity by grafting the SMDC onto an IgG1-Fc

fragment, leading to the novel Fc-SMDC format. The SMDC

and Fc-SMDC products were then compared in the in vitro

and in vivo settings.

In terms of the in vitro cytotoxic activity, both SMDC 1

IC₅₀0.863 nM) and Fc-SMDC 2 (IC₅₀15.2 nM) out-

performed unconjugated α-amanitin (IC 476 nM) and stood

out as having selective activity in a low nanomolar range of

concentration (Figure 3). The low in vitro activity displayed by

unconjugated α-amanitin is related to its hydrophilic properties

and the inability to passively penetrate the cell membrane in

prolongation of half-life. Our strategy resembles available

preclinical studies that have demonstrated that clearance of

therapeutic peptides fused to an IgG-Fc domain is increased

14- to 24-fold in FcRn-knockout mice compared to wild-type

35,36

animals.

A well-known organ of target toxicity for α-amanitin is the

liver. We reasoned that the slight liver uptake of conjugates

observed in in vivo studies (Figure 4) might be related to the

OATP1B3 receptor expressed in liver sinusoids known to be

responsible for the α-amanitin transport into the hepato-

(

cytes. Heavy accumulation of HDP 30.2284 (1) but not

HDP 30.2972 (2) was observed in the kidneys. Accumulation

of DUPA-based molecules in this organ has been reported and

is mainly related to the reabsorption via PSMA receptors

the absence of a homing vehicle that shuttles it into cells.

124

J. Med. Chem. 2021, 64, 4117−4129

Journal of Medicinal Chemistry

Article

expressed in the proximal tubules of the kidney. Based on

40:40:20; ﬂow rate: 0.25 mL/min). HRESI-MS studies were

performed at Immundiagnostik AG (Bensheim, Germany) using an

Orbitrap Elite mass spectrometer interfaced with an Ion Max Source

literature reports and biodistribution data of HDP 30.2284

1), the kidneys were identiﬁed as the organ of primary toxicity

(

HESI-II probe) (Thermo Scientiﬁc) via reverse-phase liquid

for this molecule. Grafting the DUPA-based SMDC onto the

Fc scaﬀold allowed for not only prolonging the half-life of the

conjugate but also signiﬁcantly limiting the kidney uptake and

avoiding accumulation of the toxin in this organ. Diﬀerent

proﬁles of toxin distribution were observed for the two

conjugates investigated in this study. Accumulation of α-

amanitin in the tumor over time was observed only after

administration of HDP 30.2972 (2) but not following

administration of HDP 30.2284 (1). These results indicate

that only the Fc-SMDC targets the toxin mainly to the tumor

where the conjugate is internalized, and the payload is released,

leading to a long-lasting antitumor eﬀect.

chromatography (RP-LC) gradient separation. Deconvolution of

isotopically unresolved spectra was performed with the ReSpect

algorithm using Biopharma Finder 3.0 software (Thermo Scientiﬁc).

Analytical thin-layer chromatography (TLC) was performed on

POLYGRAMSIL G/UV polyester precoated plates (40 × 80 mm ,

0.20 mm silica gel 60), and compound visualization was accomplished

with UV light and ninhydrin or Vaughn’s staining reagents. Flash

chromatography was carried out using a Teledyne ISCO CombiFlash

RF system on Silica RediSep Rf disposable columns. Solid-phase

peptide synthesis (SPPS) was performed using the automated

microwave peptide synthesizer CEM Liberty Blue or the manual

microwave peptide synthesizer CEM Discover System. All peptides

and conjugates were puriﬁed by preparative RP-HPLC (VWR LaPrep

Sigma LP1200 pumps, VWR LaPrep Sigma 3101 UV detector;

Compared to SMDC (1), the chemically programmed and

armed Fc-SMDC (2) improved the therapeutic precision and

allowed for eﬃcient toxin delivery in vivo selectively to cells

expressing the targeted PSMA receptor, as conﬁrmed in the in

vivo eﬃcacy study (Figure 5). Only Fc-SMDC (2) allowed for

obtaining dose-dependent and long-term in vivo antitumor

activity. Our results resemble the ﬁnding in the ﬁeld of ADCs,

showing that for the cytotoxic payload, a certain intratumoral

threshold concentration has to be reached to support sustained

column: Phenomenex Luna 10 μm C18(2) 100 Å, 250 × 21.2 mm )

and analyzed by analytical RP-HPLC (VWR Hitachi Chromaster

5110 binary HPLC pump, VWR Hitachi Chromaster 5430 diode

array detector (DAD); column: Phenomenex Luna 10 μm C18(2)

00 Å, 250 × 4.6 mm ). Fc protein was puriﬁed by Protein A-based

aﬃnity chromatography (Bio-rad NGC 100 Medium-Pressure

Chromatography system; column: Tosoh Bioscience ToyoScreen

AF-rProtein A HC-650F 5 mL) and dialyzed in Thermo Fisher

Scientiﬁc Slide-A-Lyzer cassettes (MWCO 20 000, 12−30 mL). Fc-

conjugates were puriﬁed by preparative size-exclusion fast-protein

eﬃcacy. Additionally, the proof-of-concept study presented

herein demonstrates that for conjugates based on hydrophilic

payloads like α-amanitin, a prolonged half-life is needed to

allow for gradual toxin accumulation in the tumor and achieve

a long-lasting antitumor response.

liquid chromatography (SEC-FPLC, AKTA Start system, HiLoad 26/

600 Superdex 200 pg column or HiLoad 16/600 Superdex 200 pg

column) and analyzed by analytical SEC (Knauer PLATINblue

HPLC with DAD, column: Tosoh Bioscience TSKgel UP-SW3000, 2

μm, 4.6 mm ID × 30 cm). Protein and protein-conjugates were

concentrated using Amicon Ultra-15 Centrifugal Filters MWCO

CONCLUSIONS

■

50 000 (Millipore) and ﬁltered through disposable 0.22 μm sterile

In the present study, we reported for the ﬁrst time the

conjugation of a SMDC onto a human IgG1-Fc fragment. With

a size approximately equal to 40% of conventional ADCs, the

novel platform merges the favorable properties of small-sized

therapeutics with the PK properties of an ADC.

Millex-GV syringe ﬁlters (Millipore). Concentration measurements

were carried out using a Thermo Fisher Scientiﬁc NanoDrop 2000c

spectrophotometer (λ = 280, 310 nm, sample size 3 μL). SDS-PAGE

analysis was performed on 4−20% precast polyacrylamide gels (Mini-

PROTEAN TGX precast protein gel, Bio-rad) in Mini-PROTEAN

electrophoresis cells (Bio-rad). Marker PageRuler Plus Prestained

Protein Ladder was purchased from Thermo Scientiﬁc. Western blot

analyses were performed using a primary rabbit α-amanitin polyclonal

antiserum (produced at Heidelberg Pharma Research GmbH) probed

with an antirabbit IgG-HRP-linked antibody (Cell Signaling

Technology). For chemoluminescence detection, Clarity Western

ECL substrates (Bio-rad) were used.

General Syntheses. The synthesis route for α-amanitin

derivatives 12 and 13 is fully shown in Scheme S1 . Starting from

natural α-amanitin (9), reaction with bromide (10) followed by

deprotection gave the intermediate S-1. The title derivatives were

prepared by condensation of the intermediate S-17 with the

appropriate NHS ester-activated cross-linker.

We demonstrated that, in contrast to the majority of

SMDCs that utilize more hydrophobic payloads that are able

to passively penetrate tumor cells, SMDCs featuring a

hydrophilic payload like α-amanitin are not a suitable format

for development of therapeutics for solid-tumor therapy.

Prolongation of α-amanitin based SMDSs half-life is necessary

to ensure exposure of the target tissue to the hydrophilic drug

in the conjugated form, allowing for its gradual uptake and

accumulation in the tissue.

The two-step strategy applied here to assemble the Fc-

SMDC might become a general approach for the chemical

programming and arming of an Fc scaﬀold. The single generic

Fc-LPETGG fragment might be indeed programmed against a

variety of targets and be accessible even to chemically

synthesized ligands not generally amenable to genetic fusion.

Also, such a strategy oﬀers the opportunity to combine

diﬀerent targeting ligands with a variety of linkers, toxic

warheads, and conjugation chemistries, thereby expanding the

landscape of tumor-targeted technologies.

SMDC (1). Compound 8 (12.97 mg, 0.015 mmol) was dissolved in

DMSO (1 mL). A solution of 13 (21.03 mg, 0.015 mmol) in DMSO

(

2 mL) was added at room temperature under argon. DIPEA (5.15

μL, 0.03 mmol) was added undiluted. The reaction mixture was

stirred at room temperature for 20 h and then puriﬁed by preparative

RP-HPLC [λ = 305 nm; gradient: 0−5 min 5% B; 20−25 min 100%

B; 27−35 min 5% B; A = water with 0.05% TFA; B = MeOH with

.05% TFA]. Solvents were evaporated and SMDC 1 was freeze-dried

overnight from BuOH:H O (4:1, v−v; 4 mL) and isolated as a

colorless powder (14.53 mg, 43%). MS (ESI): m/z calcd for

EXPERIMENTAL SECTION

■

C H N O S + 2Na : 1146.2, found: 1146.5 [M + 2Na] .

96 124 20 32 2

General Information. Solvents and reagents were purchased

from commercial vendors and used without further puriﬁcation. ESI-

MS studies were performed using a Thermo Orbitrap LTQ XL mass

spectrometer (Thermo Scientiﬁc) connected to an Agilent 1200 series

high-performance liquid chromatography (HPLC) system (isocratic

mode; mobile phase composition: methanol/acetonitrile/water,

Retention time, 13 853 min; HPLC purity, 93.7%.

Fc-SMDC (2). DBCO-amanitin precursor 12 (20 equiv, 12.18 mg)

was dissolved in ACN/H O (3:1, 1.28 mL) and added to DUPA-Fc

15 (24 mg, 48.6 μM) in PBS buﬀer (pH 7.4, 8.46 mL). DMSO (2.24

mL) was added and the mixture was incubated at 37 °C for 24 h.

Puriﬁcation was performed by SEC-FPLC. The conjugate was

125

J. Med. Chem. 2021, 64, 4117−4129

Journal of Medicinal Chemistry

Article

concentrated to a ﬁnal volume of 7.5 mL and ﬁltered through a 0.22

μm sterile ﬁlter prior to its use in biological assays. The concentration

of conjugate 2 was determined as 3.16 mg/mL (23.7 mg) by Abs

for C H N O S + H : 859.98 [M + H] ; found: 859.33; calcd for

40 54 6 13

C H N O S + Na : 881.96 [M + Na] ; found: 881.33.

Boc-Val-Ala-PAB-α-amanitin (11). Vacuum-dried α-amanitin (57

mg, 0.062 mmol) was dissolved in dry dimethyl acetamide (DMA; 3

mL) under argon at room temperature. Boc-Val-Ala(SEM)-PAB-Br

linker 10 (145.5 mg, 0.248 mmol) and 0.2 M solution of cesium

carbonate (Cs CO ) (372.2 μL, 0.074 mmol) were added. After 4 h at

80 nm

MW = 61425.21 Da, ε₂₈₀= 85 500 cm⁻

−11

). Retention time, 9.7

(

min; SEC-HPLC purity, >99%.

-Benzyl 1-(tert-butyl) (((S)-1,5-di-tert-butoxy-1,5-dioxopentan-

-yl)carbamoyl)-L-glutamate (5). α,γ-Di-tert-butyl L-glutamate (2 g,

.76 mmol) was added in portions at 0 °C to a solution of

room temperature, the reaction mixture was acidiﬁed to pH 5 with

AcOH. The solvent was removed in vacuo, and the residue was

puriﬁed by RP-HPLC (λ = 305 nm; gradient: 0−5 min 5% B; 20−25

min 100% B; 27−35 min 5% B; A = water; B = MeOH). The solvents

disuccinimidyl carbonate (DSC) (1.73 g, 6.76 mmol) in N,N-

dimethylformamide (DMF) (31.6 mL) to form the NHS-activated

ester 4. After 50 min, triethylamine (TEA) (937 μL, 6.76 mmol) was

added. After complete conversion, α-tert-butyl-γ-benzyl L-glutamate

were then evaporated to dryness, aﬀording 11 as a colorless solid

(2.23 g, 6.76 mmol) and TEA (1.87 mL, 13.52 mmol) were added at

(54.46 mg, 62%). MS (ESI): m/z calcd for C65

1425.70 [M + H] ; found: 1425.23.

19SSi + H :

°C. The reaction mixture was stirred overnight at room

temperature. DMF was removed in vacuo and the residue was

dissolved in methyl t-butyl ether (MTBE) (100 mL). The organic

layer was washed with a 15% citric acid solution (2 × 100 mL), water

DBCO-Val-Ala-PAB-O-α-amanitin (12). α-Amanitin precursor S-

7 (80.32 mg, 0.067 mmol) was dissolved in absolute DMF (1.6 mL).

Dibenzocyclooctine-N-succinimidyl ester (DBCO-SE) (29.8 mg,

.074 mmol) dissolved in DMF (1.6 mL) and DIPEA (22.9 μL,

.13 mmol) was added to the solution. The reaction mixture was

(2 × 100 mL), saturated NaHCO solution (2 × 100 mL), and water

(80 mL) in sequence. The organic layer was dried over MgSO₄,

stirred at rt for 2.5 h.

ﬁltered, and concentrated. The resulting yellowish oil was puriﬁed by

chromatography on a silica gel column with a gradient of 0−33%

EtOAc in hexane to provide urea 5 as a colorless syrup (3.02 g, 77%).

The reaction was quenched by adding H O (100 μL), and DMF

was evaporated in vacuo. The residue was dissolved in methanol

(MeOH) (2 mL) and dripped into precooled MTBE (40 mL) and

centrifuged at 0 °C. The pellet was washed with MTBE (40 mL),

collected, and dried in vacuo. The compound was puriﬁed by RP-

HPLC [λ = 305 nm; gradient: 0−15 min 5% B; 18 min 100% B; 1.5−

MS (ESI): m/z calcd for C H N O + H : 579.72 [M + H] ; found:

79.17; calcd for C H N O + Na : 601.70 [M + Na] ; found:

01.35; calcd for C H N O + Na : 1180.41 [2M + Na] ; found:

30 46 2 9

180.35.

S)-5-(tert-Butoxy)-4-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-

-yl)ureido)-5-oxopentanoic Acid (6). Compound 5 (3.02 g, 5.21

22 min 5% B; A = water with 0.05% TFA, B = ACN; ﬂow rate: 30

(

mL/min]. Fractions corresponding to the product were directly

lyophilized aﬀording 12 (77.88 mg, 78%) as a white powder. MS

mmol) was hydrogenated at room temperature in ethyl acetate

EtOAc) (27.3 mL) and in the presence of Pd-C overnight. Palladium

(

ESI): m/z calcd for C H N O S + H : 1482.67 [M + H] ; found:

(

73 88 14 18

481.42; calcd for C H N O S + 2H : 741.84 [M + 2H] ;

was ﬁltered oﬀ, and the ﬁltrate was washed thoroughly with EtOAc.

The ﬁltrate was concentrated under reduced pressure to provide

DUPA precursor 6 as a clear colorless syrup (2.45 g, 96%). MS (ESI):

m/z calcd for C H N O + H : 489.59 [M + H] ; found: 489.20;

calcd for C H N O + Na : 978.16 [2M + Na] ; found: 978.22.

73 88 14 18

found: 741.42.

Maleimidocaproyl-Val-Ala-PAB-α-amanitin (13). NH -Val-Ala-

PAB-α-amanitin 17 (10.0 mg, 0.0076 mmol) was dissolved in dry

DMF (200 μL). ECMS (4.69 mg, 0.015 mmol), dissolved in DMF

(104 μL), and DIPEA (5.18 μL, 0.0304 mmol) were added. After 1 h

DUPA-8-Aoc-Phe-Phe-Cys-OH (8). Cys-preloaded 2-chlorotrityl

at room temperature under argon, the mixture was dripped into

precooled MTBE (40 mL) and centrifuged at 0 °C. The precipitate

was collected, washed with MTBE (40 mL), centrifuged, and

collected. The crude product was dried in vacuo and puriﬁed by

RP-HPLC [λ = 305 nm; gradient: 0−5 min 5% B; 20−25 min 100%

B; 27−35 min 5% B; A = water with 0.05% TFA; B = MeOH with

(H-Cys(Trt)-2ClTrt) resin (391 mg, 0.25 mmol) was swollen in

dimethylformamide (DMF)/dichloromethane (DCM) (1:1) for 30

min prior to use. For each coupling, 2.5 equiv of Fmoc-protected

amino acid or 6, 4.25 equiv of 3-[bis(dimethylamino)methyliumyl]-

H-benzotriazol-1-oxide hexaﬂuorophosphate (HBTU), 4.25 equiv of

N-hydroxybenzotriazole (HOBt), and 8.5 equiv of N-ethyl-N-

propan-2-yl)propan-2-amine (DIPEA) were used. Each coupling

.05% TFA]. Upon freeze-drying, the title compound was obtained as

(

a colorless powder (4.22 mg, 40%). MS (ESI): m/z calcd for

C H N O S : 1387.52 [M] ; found: 1387.42; C H N O S +

reaction was carried out at 60 °C and 40 W for 10 min. After each

coupling reaction, the resin was washed with DCM (×3) and DMF

86 14 19

64 86 14 19

H : 694.79 [M + 2H] ; found: 694.33; C H N O S + H + K :

13.83 [M + H + K] ; found: 713.33.

DUPA-8-Aoc-Phe-Phe-(EG ) -Orn(N )-Lys(Gly )-NH (15). Amphi-

64 86 14 19

(

×3) in sequence. Then, 20% piperidine in DMF was added to the

reaction vessel and two deprotection cycles (60 °C, 40 W, 30 s; 60

C, 40 W, 2.5 min) were performed. After draining, the resin was

Spheres 40 RAM resin (703 mg, 0.267 mmol) was swollen 1 h in

DCM, washed with, and resuspended in DMF for 30 min. The resin

washed with neat DMF (×3) and DCM (×2). Resin-bound peptide

was cleaved with a triﬂuoroethanol (TFE)/acetic acid (AcOH)/DCM

was deprotected with 20% piperidine in DMF (30 s, rt a

0 °C) and then shaken with Fmoc-Lys(Mtt)-OH (4.0 equiv), TBTU

3.99 equiv), and DIPEA (8.0 equiv) in DMF (8 mL) for 1 h at rt and

2 min, 30 W,

(

1:1:8, 10 mL) mixture at 23 °C for 1.5 h. The resin was then washed

with fresh TFE/AcOH/DCM (1:1:8) mixture (10 mL, 2 min), DCM

10 mL, 2 min), and methanol (MeOH) (10 mL, 2 min) in sequence.

(

then under MW irradiation (30 W, 50 °C, 3 min). Coupling was

repeated twice with several DMF washings in between. Fmoc was

removed by suspending the resin in 20% piperidine in DMF (3 mL)

under the conditions described above. Each coupling was then

performed by shaking the resin with the Fmoc-protected amino acid

The ﬁltrates were collected and concentrated in vacuo. The peptide

was subsequently treated with a triﬂuoroacetic acid (TFA)/

triisopropylsilane(TIS)/H O (95:2.5:2.5, 8 mL) and 1,4-dithiothrei-

tol (DTT) (362 mg) mixture and stirred at room temperature under

argon for 1.5 h. The mixture was co-evaporated with toluene (2 × 8

mL). Addition of precooled MTBE (40 mL) caused peptide

precipitation. The precipitate was isolated by centrifugation at 0 °C,

collected, and washed with additional precooled MTBE (40 mL);

centrifuged at 0 °C; and collected. The pellet was dissolved in

(

4.0 equiv), TBTU (3.99 equiv), and DIPEA (8.0 equiv) in DMF (8

mL) under MW irradiation (30 W, 50 °C, 3 min, ×3), followed by

Fmoc-removal with the conditions mentioned herein. The protected

DUPA reagent 6 (3.0 equiv) was coupled using TBTU (2.99 equiv)

and DIPEA (6.0 equiv) under MW irradiation (30 W, 50 °C, 3 min,

×3). Prior to cleavage, Mtt was removed by suspending the resin-

bound peptide in DCM/TIS/TFA (97:2:1, 4 mL) and shaking at rt

for 10 min. The procedure was repeated till no Mtt-OH could be

detected in the ﬁltrate by HPLC (approx. 20 cycles). Afterward,

acetonitrile (ACN)/H O (1:1, v:v, 2 mL) and puriﬁed in portions by

preparative RP-HPLC [λ = 210 nm; gradient: 0 min 5% B; 15−18

min 100% B; 18.50−22 min 5% B; A = water with 0.05% TFA, B =

acetonitrile; ﬂow rate: 30 mL/min]. Fractions corresponding to the

target compound were combined, evaporated, and lyophilized

coupling with Fmoc-Gly -OH (4.0 equiv), TBTU (3.99 equiv), and

DIPEA (8.0 equiv) in DMF (8 mL) was carried out under MW

overnight from tert-butanol ( BuOH)/H O (4:1, v:v, 5 mL) to aﬀord

reagent 8 as a white powder (122.9 mg, 85%). MS (ESI): m/z calcd

irradiation (30 W, 50 °C, 3 min, ×3), followed by Fmoc-removal with

126

J. Med. Chem. 2021, 64, 4117−4129

Journal of Medicinal Chemistry

Article

the conditions mentioned herein. The resin was then extensively

washed with DCM and dried in vacuo. The peptide was cleaved from

Three animals per group were injected with a doubling increasing

dose from a dose with no eﬀect until clinical signs such as net body

weight loss of more than 20%; lack of recovery; and/or one of the

following symptoms of lack of motility, hind leg paralysis, cachexia,

poor general health condition, or general signs of pain occurred.

In Vivo Eﬃcacy Studies. 6−8 week old CB17-Scid male mice

the resin and totally deprotected with a TFA/anisole/TIS/H O

(

94:2:2:2, 20 mL) cocktail for 2 h at rt. The mixture was precipitated

in four portions in precooled MTBE (40 mL/each), and the pellets

were collected by centrifugation at 0 °C for 10 min. The pellets were

collected, dried in vacuo, and dissolved in ACN/H O (1:1, v−v) for

animals were subcutaneously inoculated with 2.5 × 10 LNCaP tumor

puriﬁcation by RP-HPLC [λ = 210 nm; gradient: 0 min 5% B; 14 min

cells in 200 μL of a 1:1 mixture of red phenol-free RPMI (Gibco) and

Matrigel (Corning) into the right ﬂank. The therapy was started once

0% B; 19 min 45% B; 20−21 min 100% B; 22 min 5% B; A = water

with 0.05% TFA, B = ACN; ﬂow rate: 30 mL/min]. The desired

a mean tumor volume reached approx. 100 mm . The compounds

compound was directly lyophilized aﬀording 15 as a white powder

were administered at doses and frequencies based on the ﬁndings of

toxicity and pharmacokinetic studies. Tumors were measured using

external calipers at least twice per week.

In Vivo Pharmacokinetic and Biodistribution Study. LNCaP

tumor-bearing CB17-Scid male mice were injected with 4× MTDs of

HDP 30.2284 (0.184 mg/kg) and 1× MTD HDP 30.2972 (1.0 mg/

kg). The animals were sacriﬁced at predeﬁned time-points. The

serum, tumor, kidney, and liver were isolated, snap-frozen in liquid

nitrogen, and stored at −70 °C.

Competitive Anti-α-amanitin ELISA. Approximately 100 mg of

tissue was transferred to a FastPrep tube (MP Biomedicals). A triple

volume of mixed gender mouse serum (Seralab) with respect to the

mass of the organ was added to the tube. One steel grinding ball (MP

Biomedicals) was added to each tube, and the samples were

homogenized using a planetary ball mill (Precellys 24 Tissue

Homogenizer, Bertin Instruments). The homogenate was centrifuged

for 5 min at 14 000 rpm and 4 °C (Thermo Fischer Scientiﬁc,

Tabletop centrifuge FRESCO 17), and the supernatants were

collected. For competitive α-amanitin ELISA, 60 μL of the tissue

supernatant or 60 μL of the mouse serum collected in the

pharmacokinetic study was precipitated with 240 μL of 100% ethanol

and incubated for 20 min at −20 °C to ensure complete protein

precipitation. After centrifugation, the supernatants were collected

and evaporated using a rotational vacuum concentrator (Martin

Christ GmbH) for 3 h at 1300 rpm and 53 °C. For determination of

standard curves, reference solutions of α-amanitin and HDP 30.2284

ranging from 0.4 to 8100 nM were prepared in the mouse serum. The

mouse serum containing the highest DMSO concentration was used

as the blank. Then, 60 μL of the standard solutions and the blank

were precipitated and processed as described above.

−

(

119.46 mg, 28%). MS (ESI): m/z calcd for C H N O − H :

113 17 24

−

599.79 [M − H] ; found: 1598.83; calcd for C H N O

−

113 17 24

−

2−

H : 799.39 [M − 2H] ; found: 799.00.

DUPA-Pep-Fc (16). Fc-LPETGG 14 (40 mg, 20.65 μM) was mixed

with the peptide sequence 15 (50 equiv, 1 mM) in SrtA reaction-

buﬀer (Tris-HCl 50 mM pH 7.5, NaCl 150 mM, CaCl 5 mM) in the

presence of a SrtA pentamutant (eSrtA) enzyme (0.125 equiv, 2.6

μM). The reaction was allowed to proceed for 18 h at 25 °C and then

puriﬁed using SEC-FPLC to remove eSrtA and excess of the peptide.

The column was ﬁrst equilibrated with PBS buﬀer (pH 7.4), and then

DUPA-Fc 16 was eluted using the same buﬀer as used for column

equilibration. The ﬂow-through from the column was concentrated

and ﬁltered through a sterile ﬁlter. The concentration of DUPA-Fc

conjugate 16 was determined as 3.6 mg/mL (27.87 mg) by Abs

80 nm

−11

(

MW = 58461.89 Da, ε₂₈₀= 74675.1 cm⁻¹

min; SEC-HPLC purity, 99%.

). Retention time, 9.4

NH -Val-Ala-PAB-α-amanitin (17). Boc-Val-Ala-PAB-α-amanitin

1 (134.29 mg, 0.0943 mmol) was dissolved in TFA (4.98 mL) and

stirred at room temperature for 2 min and then concentrated to

dryness. The residue was dissolved in water (4.98 mL), and the pH

was adjusted to 10 with a 3.2% NH₃aqueous solution added

dropwise. The resulting suspension was freeze-dried and the resulting

powder was puriﬁed by preparative RP-HPLC (λ = 305 nm; gradient:

−2 min 5% B; 2−10 min 20% B; 10−10.5 min 25% B; 10.5−13 min

100% B; 13−14 min 5% B; A = water with 0.05% TFA; B = ACN).

Upon elution, the title compound was directly freeze-dried overnight

and obtained as a colorless powder (68.59 mg, 55%). MS (ESI): m/z

calcd for C H N O S + H : 1194.53 [M + H] ; found: 1194.8;

75 13 16

calcd for C H N NaO S + Na : 1216.51 [M + Na] ; found:

75 13

217.8.

ELISA plates were coated overnight with 50 μL of the anti-α-

amanitin capture serum (Polyclonal rabbit, Heidelberg Pharma

Research GmbH), c = 6.67 μg/mL diluted in PBS. The plates were

blocked with 200 μL/well ELISA blocking buﬀer (3% BSA/PBS-

solution) for 1 h at 100 rpm and 37 °C and washed with 300 μL/well

wash buﬀer (0.05% Tween/PBS) using an automated Hydrospeed

ELISA Washer (Tecan). Evaporated samples prepared as described

above were reconstituted in the ELISA sample buﬀer (1% BSA/PBS

with 20% EtOH) and mixed with 50 μL of 1 nM biotin-α-amanitin

conjugate (Heidelberg Pharma Research GmbH) also reconstituted in

the ELISA sample buﬀer. Then, 50 μL of the 1:1 mixture of sample

and biotinylated α-amanitin was applied onto the ELISA plate in

duplicate and incubated for 1 h at 37 °C. The plate was washed three

times with 300 μL of the ELISA wash buﬀer using an automated

Hydrospeed ELISA Washer (Tecan). Subsequently 50 μL of

streptavidin-HRP (1 μg/mL) was added in each well and incubated

for 1 h at 100 rpm and 37 °C. After washing, 100 μL of 0.1 mg/mL

TMB reagent reconstituted in 0.1 M sodium acetate buﬀer containing

0.001% H O was added. After 15 min, the reaction was stopped with

In Vitro Cytotoxicity Assay. LNCaP cells were cultivated in

RPMI medium and PC3 cells in DMEM (PAN-Biotech GmbH)

supplemented with 10% fetal bovine serum L-glutamine, 100 units of

penicillin/mL, and 100 μg/mL streptomycin. Cells were incubated at

7 °C in 100% humidity and 5% CO saturation. For the cytotoxicity

assay, 2 × 10 cells/well were plated in 96-well black clear bottom

plates (PerkinElmer) in 90 μL of the medium and allowed to attach

for 16 h. A panel of eight serial dilutions of the test compounds was

prepared in cell culture media, and 10 μL was added to each well and

incubated for an additional 96 h. Next, 100 μL of CellTiterGlo 2.0

reagent (Promega) was added directly to the cell culture media and

incubated for 10 min to allow for cell lysis. Luminescence signal

intensities were measured using a microplate reader (Fluostar Optima

BMG Labtec). The background was determined from wells

containing the medium only with CellTiterGlo 2.0 reagent and

subtracted from each value. In vitro competitive cytotoxic assay was

performed in the presence of 100 × molar excess of 2-PMPA over

HDP 30.2284 (SMDC) and 200 × molar excess of 2-PMPA over

HDP 30.2972 (Fc-SMDC).

50 μL of 1 M H SO . Absorbance was measured at 450 nm and

Determination of the Maximum Tolerated Dose. All

experiments were carried out following the guidelines of the German

Animal Welfare Act and all relevant laws and regulations. Protocols

corrected with the background absorbance at 570 nm (Fluostar

Optima BMG Labtec). The concentration of α-amanitin-containing

compounds was calculated based on the interpolation of the sigmoidal

standard curve using GraphPad Prism 7.0 software, and for organs

and tumor, the calculation was based on the mass of the analyzed

tissue.

Sandwich ELISA for Determination of HDP 30.2972

Concentration. ELISA was performed as described above with the

following changes. HDP 30.2972 standards were prepared in the

sample buﬀer (Candor Biosciences GmbH) as 1:2 serial dilutions

were approved by the local regulatory authority (Regierungsprasidium

Karlsruhe, Germany, ﬁle numbers: 35-9185.82/A-22/15, 35-9185.81/

G-197/15, 35-9185.81/G-13/16, and 35-9185.81/G-157/17). The

tolerability of compounds was tested in 6−8 week old CB17-Scid

male mice. The conjugates were diluted in PBS (pH 7.4) containing a

maximal concentration of 5% DMSO. Survival and clinical signs were

determined daily. The body weight was determined twice a week.

127

J. Med. Chem. 2021, 64, 4117−4129

Journal of Medicinal Chemistry

https://pubs.acs.org/10.1021/acs.jmedchem.1c00003

Article

ranging from 1.6 to 400 pM. Standards were analyzed in parallel with

the serum and organ extracts. Sera and organ extracts were diluted in

the sample buﬀer (Candor Biosciences GmbH) in two steps, (1)

Author Contributions

:100 and (2) 1:10, to reach a ﬁnal dilution of 1:1000. Next, 50 μL of

F.G. and B.K. contributed equally to this work. All authors

each sample was applied in duplicate on the ELISA plate coated with

α-amanitin capture serum and incubated at 37 °C for 1 h on an orbital

microplate shaker at 100 rpm. After washing 50 μL of a 1:10 000

dilution of secondary antibody rabbit anti-Human-IgG-HRP (Abcam

ab98576) reconstituted in sample buﬀer was added per well and

incubated for 1 h, 100 rpm, at 37 °C.

have given approval to the ﬁnal version of the manuscript.

Notes

The authors declare the following competing ﬁnancial

interest(s): A.P. is a full-time employee of Heidelberg Pharma

AG, F.G., B.K., T.H., and C.M. are full-time employees of

Heidelberg Pharma Research GmbH. Heidelberg Pharma

Research GmbH is a German biotech company operating in

the ﬁeld of antibody-drug conjugates and is a daughter

company of Heidelberg Pharma AG. A.P. and T.H. are

shareholders of Heidelberg Pharma AG.

Statistical Analysis. The sample size was empirically set at n = 3

for in vitro cell experiments, n = 3 for in vivo biodistribution and

pharmacokinetic studies, and n = 8−10 for in vivo eﬃcacy studies.

Determination of IC values was done using GraphPad Prism 7.0

software. The Mann−Whitney test was used to compare the diﬀerent

experimental arms in vivo. The level of signiﬁcance was set at values

P < 0.05, **P < 0.01, and ***P < 0.001. The concentration of

compounds was calculated based on the interpolated standard curves.

The half-lifetime of small molecular conjugate HDP 30.2284 was

calculated using a one-phase decay exponential nonlinear regression

curve ﬁt. The half-lifetime of HDP 30.2972 (Fc-SMDC) was

calculated based on a two-phase decay nonlinear regression curve ﬁt.

ACKNOWLEDGMENTS

■

F.G. and B.K. acknowledge the European Union within the

H2020 Marie Skłodowska-Curie European Training Network

MAGICBULLET (MSCA-ITN-2014-ETN, grant agreement

number 642004).

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00003.

■

ABBREVIATIONS

SMDC, small-molecule drug conjugate; ADC, antibody drug

conjugate; MTD, maximum tolerated dose

■

Supplementary materials and methods; general synthesis

of α-amantin derivatives 12 and 13; synthesis of Boc-

Val-Ala(SEM)-PAB-Br linker 10; cloning of plasmid for

protein expression; expression and puriﬁcation of

protein Fc-LPETGG (14); and production of eSrtA

enzyme; Supplementary ﬁgures: Figure S1: RP-HPLC

trace of pure SMDC (1); Figure S2: SEC-HPLC trace of

puriﬁed DUPA-Fc (16); Figure S3: SEC-HPLC trace of

puriﬁed DUPA-Fc-amanitin HDP 30.2972 (2); Figure

S4: Cytotoxicity of DUPA-alpha-amanitin SMDC HDP

REFERENCES

■

(

1) Abdollahpour-Alitappeh, M.; Lotfinia, M.; Larki, P.; Faghfourian,

B.; Sepehr, K. S.; Abbaszadeh-Goudarzi, K.; Abbaszadeh-Goudarzi,

G.; Johari, B.; Zali, M. R.; Bagheri, N.; et al. Antibody-drug conjugates

(ADCs) for cancer therapy: Strategies, challenges, and successes. J.

Cell. Physiol. 2019, 234, 5628−5642.

(2) Amiri-Kordestani, L.; Blumenthal, G. M.; Xu, Q. C.; Zhang, L.;

Tang, S. W.; Ha, L.; Weinberg, W. C.; Chi, B.; Candau-Chacon, R.;

Hughes, P.; Russell, A. M.; Miksinski, S. P.; Chen, X. H.; McGuinn,

W. D.; Palmby, T.; Schrieber, S. J.; Liu, Q.; Wang, J.; Song, P.;

Mehrotra, N.; Skarupa, L.; Clouse, K.; Al-Hakim, A.; Sridhara, R.;

Ibrahim, A.; Justice, R.; Pazdur, R.; Cortazar, P. FDA approval: ado-

trastuzumab emtansine for the treatment of patients with HER2-

positive metastatic breast cancer. Clin. Cancer Res. 2014, 20, 4436−

0.2284; Fc analogue HDP 30.2972 and unconjugated

alpha-amanitin in PSMA negative (PSMA -) PC3 cell

line (PDF)

441.

(

3) Tsumura, R.; Manabe, S.; Takashima, H.; Koga, Y.; Yasunaga,

AUTHOR INFORMATION

Corresponding Author

Andreas Pahl − Heidelberg Pharma Research GmbH,

Heidelberg Pharma AG, 68526 Ladenburg, Germany;

orcid.org/0000-0002-6294-0335; Email: andreas.pahl@

hdpharma.com

■

M.; Matsumura, Y. Influence of the dissociation rate constant on the

intra-tumor distribution of antibody-drug conjugate against tissue

factor. J. Controlled Release 2018, 284, 49−56.

(4) Leamon, C. P.; Reddy, J. A.; Vetzel, M.; Dorton, R.; Westrick, E.;

Parker, N.; Wang, Y.; Vlahov, I. Folate targeting enables durable and

specific antitumor responses from a therapeutically null tubulysin B

analogue. Cancer Res. 2008, 68, 9839−9844.

Authors

(5) Kularatne, S. A.; Wang, K.; Santhapuram, H. K.; Low, P. S.

Prostate-specific membrane antigen targeted imaging and therapy of

prostate cancer using a PSMA inhibitor as a homing ligand. Mol.

Pharm. 2009, 6, 780−789.

Francesca Gallo − Heidelberg Pharma Research GmbH,

Heidelberg Pharma AG, 68526 Ladenburg, Germany

Barbara Korsak − Heidelberg Pharma Research GmbH,

Heidelberg Pharma AG, 68526 Ladenburg, Germany

(

6) Leamon, C. P.; Reddy, J. A.; Bloomfield, A.; Dorton, R.; Nelson,

M.; Vetzel, M.; Kleindl, P.; Hahn, S.; Wang, K.; Vlahov, I. R. Prostate-

Specific Membrane Antigen-Specific Antitumor Activity of a Self-

Immolative Tubulysin Conjugate. Bioconjugate Chem. 2019, 30,

Christoph Muller − Heidelberg Pharma Research GmbH,

Heidelberg Pharma AG, 68526 Ladenburg, Germany

Torsten Hechler − Heidelberg Pharma Research GmbH,

Heidelberg Pharma AG, 68526 Ladenburg, Germany

Desislava Yanakieva − Department of Biochemistry, Technical

University of Darmstadt, 64287 Darmstadt, Germany

Olga Avrutina − Department of Biochemistry, Technical

University of Darmstadt, 64287 Darmstadt, Germany

Harald Kolmar − Department of Biochemistry, Technical

University of Darmstadt, 64287 Darmstadt, Germany

805−1813.

(7) Ginj, M.; Zhang, H.; Waser, B.; Cescato, R.; Wild, D.; Wang, X.;

Erchegyi, J.; Rivier, J.; Macke, H. R.; Reubi, J. C. Radiolabeled

somatostatin receptor antagonists are preferable to agonists for in vivo

peptide receptor targeting of tumors. Proc. Natl. Acad. Sci. U.S.A.

8) Cazzamalli, S.; Corso, D.; Widmayer, A.; Neri, F. D. Chemically

006, 103, 16436−16441.

Defined Antibody- and Small Molecule-Drug Conjugates for in Vivo

Tumor Targeting Applications: A Comparative Analysis. J. Am. Chem.

Soc. 2018, 140, 1617−1621.

Complete contact information is available at:

128

J. Med. Chem. 2021, 64, 4117−4129

Journal of Medicinal Chemistry

Article

10) Molineux, G.; Newland, A. Development of romiplostim for the

9) Roopenian, D. C.; Akilesh, S. FcRn: the neonatal Fc receptor

comes of age. Nat. Rev. Immunol. 2007 , 7 , 715 −725.

(27) Dickgiesser, S.; Rasche, N.; Nasu, D.; Middel, S.; Horner, S.;

Avrutina, O.; Diederichsen, U.; Kolmar, H. Self-Assembled Hybrid

Aptamer-Fc Conjugates for Targeted Delivery: A Modular Chemo-

enzymatic Approach. ACS Chem. Biol. 2015, 10, 2158−2165.

(28) Lee, S.; Lee, Y.; Kim, H.; Lee, D. Y.; Jon, S. Bilirubin

Nanoparticle-Assisted Delivery of a Small Molecule-Drug Conjugate

for Targeted Cancer Therapy. Biomacromolecules 2018, 19, 2270−

2277.

treatment of patients with chronic immune thrombocytopenia: from

bench to bedside. Br. J. Haematol. 2010, 150, 9−20.

in Primary Immune Thrombocytopenia: A Possible Role of the Fc

Fragment of Romiplostim? Front. Immunol. 2019, 10, No. 1196.

11) Schifferli, A.; Nimmerjahn, F.; Kuhne, T. Immunomodulation

(

12) Sokoloff, R. L.; Norton, K. C.; Gasior, C. L.; Marker, K. M.;

(29) Perrino, E.; Steiner, M.; Krall, N.; Bernardes, G. J.; Pretto, F.;

Casi, G.; Neri, D. Curative properties of noninternalizing antibody-

drug conjugates based on maytansinoids. Cancer Res. 2014, 74, 2569−

2578.

(30) Dal Corso, A.; Gebleux, R.; Murer, P.; Soltermann, A.; Neri, D.

A non-internalizing antibody-drug conjugate based on an anthracy-

cline payload displays potent therapeutic activity in vivo. J. Controlled

Release 2017, 264, 211−218.

(31) Lv, Q.; Yang, J.; Zhang, R.; Yang, Z.; Yang, Z.; Wang, Y.; Xu, Y.;

He, Z. Prostate-Specific Membrane Antigen Targeted Therapy of

Prostate Cancer Using a DUPA-Paclitaxel Conjugate. Mol. Pharm.

Grauer, L. S. A dual-monoclonal sandwich assay for prostate-specific

membrane antigen: levels in tissues, seminal fluid and urine. Prostate

(

000, 43, 150−157.

13) Silver, D. A.; Pellicer, I.; Fair, W. R.; Heston, W. D.; Cordon-

Cardo, C. Prostate-specific membrane antigen expression in normal

and malignant human tissues. Clin. Cancer Res. 1997, 3, 81−85.

https://clinicaltrials.gov/ct2/show/NCT02202447.

14) Phase 1 of EC1169 In Patients With Recurrent MCRPC.

15) Petrylak, D. P.; Kantoff, P.; Vogelzang, N. J.; Mega, A.;

(

Fleming, M. T.; Stephenson, J. J., Jr; Frank, R.; Shore, N. D.; Dreicer,

R.; McClay, E. F.; Berry, W. R.; Agarwal, M.; DiPippo, V. A.;

Rotshteyn, Y.; Stambler, N.; Olson, W. C.; Morris, S. A.; Israel, R. J.

Phase 1 study of PSMA ADC, an antibody-drug conjugate targeting

prostate-specific membrane antigen, in chemotherapy-refractory

prostate cancer. Prostate 2019, 79, 604−613.

32) Cazzamalli, S.; Corso, A. D.; Neri, D. Linker stability influences

018, 15, 1842−1852.

the anti-tumor activity of acetazolamide-drug conjugates for the

therapy of renal cell carcinoma. J. Controlled Release 2017, 246, 39−

H.; Nanus, D. M.; Scher, H. I. Phase 1/2 multiple ascending dose trial

33) Moshnikova, A.; Moshnikova, V.; Andreev, O. A.; Reshetnyak,

Y. K. Antiproliferative effect of pHLIP-amanitin. Biochemistry 2013,

2, 1171−1178.

34) Bodero, L.; Lopez Rivas, P.; Korsak, B.; Hechler, T.; Pahl, A.;

(

16) Milowsky, M. I.; Galsky, M. D.; Morris, M. J.; Crona, D. J.;

George, D. J.; Dreicer, R.; Tse, K.; Petruck, J.; Webb, I. J.; Bander, N.

of the prostate-specific membrane antigen-targeted antibody drug

conjugate MLN2704 in metastatic castration-resistant prostate cancer.

Urol. Oncol. 2016, 34, 530.e15−530.e21.

(

Muller, C.; Arosio, D.; Pignataro, L.; Gennari, C.; Piarulli, U.

Synthesis and biological evaluation of RGD and isoDGR peptidomi-

metic-alpha-amanitin conjugates for tumor-targeting. Beilstein J. Org.

Chem. 2018, 14, 407−415.

(

17) Yaghoubi, S.; Karimi, M. H.; Lotfinia, M.; Gharibi, T.; Mahi-

Birjand, M.; Kavi, E.; Hosseini, F.; Sineh Sepehr, K.; Khatami, M.;

Bagheri, N.; Abdollahpour-Alitappeh, M. Potential drugs used in the

antibody-drug conjugate (ADC) architecture for cancer therapy. J.

Cell. Physiol. 2020, 235, 31−64.

(35) Wang, Y. M. C.; Sloey, B.; Wong, T.; Khandelwal, P.; Melara,

R.; Sun, Y. N. Investigation of the pharmacokinetics of romiplostim in

rodents with a focus on the clearance mechanism. Pharm. Res. 2011,

8, 1931−1938.

18) Pahl, A.; Lutz, C.; Hechler, T. Amanitins and their development

36) Wu, B.; Johnson, J.; Soto, M.; Ponce, M.; Calamba, D.; Sun, Y.

as a payload for antibody-drug conjugates. Drug Discovery Today:

Technol. 2018, 30, 85−89.

N. Investigation of the mechanism of clearance of AMG 386, a

selective angiopoietin-1/2 neutralizing peptibody, in splenectomized,

nephrectomized, and FcRn knockout rodent models. Pharm. Res.

19) Kularatne, S. A.; Zhou, Z.; Yang, J.; Post, C. B.; Low, P. S.

Design, synthesis, and preclinical evaluation of prostate-specific

membrane antigen targeted (99m)Tc-radioimaging agents. Mol.

Pharm. 2009, 6, 790−800.

37) Letschert, K.; Faulstich, H.; Keller, D.; Keppler, D. Molecular

012, 29, 1057−1065.

characterization and inhibition of amanitin uptake into human

(20) Kuester, D.; Lippert, H.; Roessner, A.; Krueger, S. The

hepatocytes. Toxicol. Sci. 2006, 91, 140−149.

cathepsin family and their role in colorectal cancer. Pathol., Res. Pract.

008, 204, 491−500.

21) Ruan, H.; Hao, S.; Young, P.; Zhang, H. Targeting Cathepsin B

for Cancer Therapies. Horiz. Cancer Res. 2015, 56, 23−40.

22) Alley, S. C.; Benjamin, D. R.; Jeffrey, S. C.; Okeley, N. M.;

(38) Chatalic, K. L.; Heskamp, S.; Konijnenberg, M.; Molkenboer-

(

Kuenen, J. D.; Franssen, G. M.; Clahsen-van Groningen, M. C.;

Schottelius, M.; Wester, H. J.; van Weerden, W. M.; Boerman, O. C.;

de Jong, M. Towards Personalized Treatment of Prostate Cancer:

PSMA I&T, a Promising Prostate-Specific Membrane Antigen-

Targeted Theranostic Agent. Theranostics 2016, 6, 849−861.

(

Meyer, D. L.; Sanderson, R. J.; Senter, P. D. Contribution of linker

stability to the activities of anticancer immunoconjugates. Bioconjugate

Chem. 2008, 19, 759−765.

(39) Zhang, D.; Dragovich, P. S.; Yu, S. F.; Ma, Y.; Pillow, T. H.;

Sadowsky, J. D.; Su, D.; Wang, W.; Polson, A.; Khojasteh, S. C.; Hop,

C. Exposure-Efficacy Analysis of Antibody-Drug Conjugates Deliver-

ing an Excessive Level of Payload to Tissues. Drug Metab. Dispos.

(

23) Thomas, J. D.; Cui, H.; North, P. J.; Hofer, T.; Rader, C.;

Burke, T. R., Jr. Application of strain-promoted azide-alkyne

cycloaddition and tetrazine ligation to targeted Fc-drug conjugates.

Bioconjugate Chem. 2012, 23, 2007−2013.

2019, 47, 1146−1155.

(

24) Swee, L. K.; Guimaraes, C. P.; Sehrawat, S.; Spooner, E.;

Barrasa, M. I.; Ploegh, H. L. Sortase-mediated modification of

α DEC205 affords optimization of antigen presentation and

immunization against a set of viral epitopes. Proc. Natl. Acad. Sci.

U.S.A. 2013, 110, 1428−1433.

(25) Wagner, K.; Kwakkenbos, M. J.; Claassen, Y. B.; Maijoor, K.;

Bohne, M.; van der Sluijs, K. F.; Witte, M. D.; van Zoelen, D. J.;

Cornelissen, L. A.; Beaumont, T.; Bakker, A. Q.; Ploegh, H. L.; Spits,

H. Bispecific antibody generated with sortase and click chemistry has

broad antiinfluenza virus activity. Proc. Natl. Acad. Sci. U.S.A. 2014,

26) Kornberger, P.; Skerra, A. Sortase-catalyzed in vitro

11, 16820−16825.

functionalization of a HER2-specific recombinant Fab for tumor

targeting of the plant cytotoxin gelonin. mAbs 2014, 6, 354−366.

129