MOLECULAR CANCER THERAPEUTICS | LARGE MOLECULE THERAPEUTICS
Design and Evaluation of Phosphonamidate-Linked
Exatecan Constructs for Highly Loaded, Stable, and
Efficacious Antibody–Drug Conjugates
Saskia Schmitt1, Paul Machui1, Isabelle Mai1, Sarah Herterich1, Swetlana Wunder1, Philipp Cyprys1,
Marcus Gerlach1, Philipp Ochtrop1, Christian P.R. Hackenberger2,3, Dominik Schumacher1, Jonas Helma1,
Annette M. Vogl1, and Marc-Andre
Kasper1
ABSTRACT
◥
Topoisomerase I (TOP1) Inhibitors constitute an emerging
payload class to engineer antibody–drug conjugates (ADC) as
next-generation biopharmaceutical for cancer treatment. Existing
ADCs are using camptothecin payloads with lower potency and
suffer from limited stability in circulation. With this study, we
introduce a novel camptothecin-based linker–payload platform
based on the highly potent camptothecin derivative exatecan. First,
we describe general challenges that arise from the hydrophobic
combination of exatecan and established dipeptidyl p-aminobenzyl-
carbamate (PAB) cleavage sites such as reduced antibody conju-
gation yields and ADC aggregation. After evaluating several linker–
payload structures, we identified ethynyl-phosphonamidates in
combination with a discrete PEG24 chain to compensate for the
hydrophobic PAB–exatecan moiety. Furthermore, we demonstrate
Introduction
With a total of 12 approved antibody–drug conjugates (ADC) up
to the present day and not less than 8 approvals by the FDA only in
the last four years, ADCs for the treatment of cancer are coming of
age (1, 2). Drug classes that are being used in those marketed ADCs
include DNA-damaging and microtubule-disrupting agents (3).
However, one class that stands out in terms of clinical efficacy
and market success is of topoisomerase I (TOP1) inhibitors, used
in trastuzumab deruxtecan (Enhertu), sacituzumab govitecan
(Trodelvy) (4) and many other preclinically and clinically inves-
tigated ADCs (5–7). Next to the application as ADC payloads, which
this publication is focusing on, camptothecin and its derivatives have
been studied for decades as small-molecule therapeutics in cancer
treatment (8).
In particular, camptothecin analogs such as DXd, a derivative of
exatecan used in Enhertu, and SN38, the active metabolite of
1
Tubulis GmbH, Planegg-Martinsried, Germany. 2Chemical Biology Department,
Leibniz-Forschungsinstitut fu€r Molekulare Pharmakologie (FMP), Berlin,
Germany. 3Department of Chemistry, Humboldt Universit€at zu Berlin, Berlin,
Germany.
S. Schmitt and P. Machui contributed equally as co-authors of this article.
Corresponding Authors: Marc-Andre
Kasper, Tubulis GmbH, Am Klopferspitz
19a, Planegg-Martinsried, Bavaria 82152, Germany. E-mail:
marc.kasper@tubulis.com; and Annette M. Vogl, annette.vogl@tubulis.com
Mol Cancer Ther 2023;XX:XX–XX
doi: 10.1158/1535-7163.MCT-23-0359
This open access article is distributed under the Creative Commons Attribution-
NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.

2023 The Authors; Published by the American Association for Cancer Research
that the identified linker–payload structure enables the construction
of highly loaded DAR8 ADCs with excellent solubility properties.
Head-to-head comparison with Enhertu, an approved camptothe-
cin-based ADC, revealed improved target-mediated killing of tumor
cells, excellent bystander killing, drastically improved linker stabil-
ity in vitro and in vivo and superior in vivo efficacy over four tested
dose levels in a xenograft model. Moreover, we show that ADCs
based on the novel exatecan linker–payload platform exhibit anti-
body-like pharmacokinetic properties, even when the ADCs are
highly loaded with eight drug molecules per antibody. This ADC
platform constitutes a new and general solution to deliver TOP1
inhibitors with highest efficiency to the site of the tumor, indepen-
dent of the antibody and its target, and is thereby broadly applicable
to various cancer indications.
irinotecan used in Trodelvy, have been contributing to the success
of the TOP1 inhibitor payload family as ADC payloads (see Fig. 1
for structures). Trodelvy has been shown to significantly prolong
overall survival (OS) compared with single-agent chemotherapy in
difficult to treat patients with estrogen receptor–positive, HER2-
negative breast cancer (9). Enhertu demonstrated superior efficacy,
with a significantly longer progression-free survival in patients with
breast cancer compared with trastuzumab-DM1 (Kadcyla) treat-
ment (10). Notably, this tremendous advantage is achieved with the
same antibody and target, only changing the linker–payload from
the tubulin-disrupting agent DM1 to a TOP1 inhibitor. Moreover,
Enhertu has been changing the paradigm of HER2-positivity,
because it showed remarkable activity in patients with HER2-low
breast cancer, a patient population previously considered as being
not amenable to HER2-based treatments (11).
Despite those remarkable achievements in the treatment of
patients with cancer with ADCs using camptothecin-based pay-
loads, there is also still room for improvement. Even though ADCs
are in theory highly targeted medications, treated patients are still
suffering from marked signs of toxicity (12) with neutropenia and
severe diarrhea being the most common adverse event leading
to dose reductions for Trodelvy and pneumonitis and interstitial
lung disease being the most common reasons for treatment dis-
continuation for Enhertu (1). Even though mechanisms of ADC-
mediated toxicities are complex and not yet fully understood, it is
discussed throughout the literature that the linker that connects the
cytotoxic drug with the antibody plays a crucial role in how efficacy
and toxicity is balanced within a given ADC (13–16). Critical
requirements on a linker include plasma stability (15, 17) and
inclusion of hydrophilicity to compensate for the typically hydro-
phobic drug to prevent undesired ADC aggregation and accelerated
plasma clearance (18, 19).
AACRJournals.org | OF1
 Schmitt et al.
Figure 1.
Overview of TOP1 inhibitor-based linker–payloads used in marketed ADCs Enhertu and Trodelvy (top), based on the payloads DXd and SN38 (2) and the structure of
the linker–payloads described herein based on exatecan with an overview over basic features of the ADCs originating from those linker systems. Payload structures
are highlighted in orange.
Despite being a highly promising ADC payload class, the conju-
gation of the camptothecin moiety, including its derivatives has been
shown to be in particular challenging in the past, especially in higher
drug-to-antibody ratios (DAR). Burke and colleagues (20) have
shown that antibody conjugates with valine–citrulline–PAB [VC–
PAB (dipeptidyl p-aminobenzyl-carbamate)]–camptothecin deriva-
tives exhibit a strong tendency to form higher molecular weight species
(HMWS). This issue could only be solved by DAR reduction
and substitution of the VC-cleavage site with a more polar glucuro-
nide (20). Similar challenges in induction of aggregation, once con-
jugated to an antibody, have been described for several exatecan-
based derivatives (21). Further improvements in the linker structure
such as the implementation of a more polar hemiaminal-based self-
immolative moiety led to the discovery of the DXd-containing linker
used in Enhertu (22, 23). This linker strategy has also been applied to
similar camptothecin analogs, all of which showing decent activity
in vitro, including bystander killing (24). In another study, hydrophilic
polysarcosins have been added to the linker to compensate for
the hydrophobic exatecan moiety (25). It should be noted that all
those examples exhibit maleimide linker chemistry for conjugation,
whereas a camptothecin-based linker–payload platform that provides
fully in vivo-stable antibody conjugates of a high DAR has still been
unprecedented until this study.
Unsaturated phosphorous(V)-based electrophiles have been
recently introduced as new cystein-selective bioconjugation
reagents for the generation of highly efficacious ADCs. We have
shown that they can easily be incorporated even into complex
molecules and they yield highly stable cysteine adducts with a high
selectivity for thiols (no modification of other amino acids than
cysteine can be observed) and fast reaction kinetics in the range of
bioorthogonal strain promoted cycloaddition reactions (26–30).
OF2 Mol Cancer Ther; 2023
Furthermore, ethynylphosphonamidates provided in combination
with solubility enhancers high DAR ADCs of hydrophobic VC–
PAB–MMAE linker–payloads without increased in vivo clear-
ance (31, 32). High DARs are important for camptothecin payloads,
in particular, because high DAR can compensate for the lower
potency compared with other ADC payloads such as auristatins or
calicheamycins (4).
Our goal in the current study was to design a novel TOP1 inhibitor-
based linker–payload platform that facilitates aggregation-free con-
struction of highly loaded DAR8 ADCs with excellent serum stability,
without enhanced in vivo clearance rates and potent and selective
in vitro and in vivo efficacy. For this, we used exatecan as payload and
conducted chemical optimization of the linker to facilitate aggrega-
tion-free ADCs even at high DARs of 8. The optimal linker–payload
structure identified carries the well-established VC–PAB cleavage site
for traceless intracellular release of the payload (33) and ethynylpho-
sphonamidates equipped with polyethylene glycol (PEG) chains of
discrete length of 24 units. The PEG chain is thereby attached in a
branched fashion to ensure close distance between antibody and
payload. We demonstrate that DAR8 ADCs based on this linker–
payload structure are superior in head-to-head comparisons with
Enhertu in a broad panel of in vitro and in vivo experiments. Moreover,
we observed excellent bystander killing as well as release of damage-
associated molecular patterns (DAMP) as an indirect measure of
immunogenic cell death (ICD) of our linker–payload, two features
that are discussed as key drivers of efficacy of ADCs (34–37). Finally,
we demonstrate excellent in vivo features of our newly identified
linker–payload structure, such as a tremendous increase in efficacy
compared with Enhertu and antibody-like pharmacokinetics (PK)
properties of the highly loaded ADCs with retained DAR8 even after
21 days of circulation.
MOLECULAR CANCER THERAPEUTICS

Materials and Methods
In vitro cytotoxicity
To investigate the cytotoxicity of the unconjugated small-molecule
TOP1 inhibitors and ADCs, respective cells were incubated for 4 days
(small molecules) and 7 days (ADCs) with increasing concentrations
of small molecules (0.015–1,000 nmol/L) and ADCs (0.05–3 mg/mL) to
generate a dose–response curve. Killing was analyzed using resa-
zurin cell viability dye at a final concentration of 55 mmol/L (Merck)
by dividing the fluorescence from control cells in medium by the
fluorescence of ADC-treated cells. Fluorescence emission at
590 nmol/L was measured on a microplate reader Infinite 200 Pro
(Tecan Group Ltd.).
Optimized procedure for the conjugation of LP5 to antibodies to
achieve DAR8
50 mL of the antibody solution at 10.0 mg/mL in P5-conjugation
buffer (50 mmol/L Tris, 1 mmol/L EDTA, 100 mmol/L NaCl, pH 8.3 at
room temperature) were mixed with 1.66 mL of a 10 mmol/L TCEP
[Tris(2-carboxyethyl)phosphine] solution (5 eq.) in P5-conjugation
buffer. Directly afterwards, 0.83 mL of a 40 mmol/L solution of LP5
dissolved in DMSO (10 eq.) were added. The mixture was shaken at
350 rpm and 25
C for 16 hours. The reaction mixtures were purified by
preparative size-exclusion chromatography (SEC) with a Superdex 200
Increase 10/300GL (Cytiva) and a flow of 0.8 mL/min eluting with
sterile PBS (Merck). The antibody-containing fractions were pooled
and concentrated by spin-filtration (Amicon Ultra 2mL MWCO:
30 kDa, Merck).
Determination of antibodies bound per cell
Antibodies bound per cell (ABC) were determined by staining of cell
lines with saturating concentrations of HER2-PE (BioLegend) or the
respective isotype control and acquisition by flow cytometry on a
CytoFLEX S flow cytometer. Quantification of ABCs as an estimation
for surface receptor expression was performed by simultaneous acqui-
sition of QuantiBRITE beads (BD Biosciences) conjugated with four
levels of PE and generation of a standard curve.
Analysis of HER2-binding of trastuzumab-LP5 and enhertu
To determine equilibrium-binding constants (KD; as an avidity
measurement), HER2-positive SKBR-3 cells were incubated with
antibodies at increasing concentrations up to saturation and stained
with an AF488-labeled goat anti-human IgG (HþL) secondary anti-
body (Thermo Fisher Scientific). Cells were acquired by flow cyto-
metry on a CytoFLEX S flow cytometer (Beckman Coulter). Mean
fluorescence intensity (MFI) ratios were determined by dividing the
MFI of the antibody-incubated cells by the fluorescence of the sec-
ondary antibody control. Data points were analyzed by a non-linear
regression using a one-site–specific binding model.
Analysis of HER2-mediated internalization of trastuzumab-LP5
and enhertu
For pHrodo-based investigation of internalization, antibodies were
labeled with the pHrodo Deep Red Antibody Labeling Kit (Thermo
Fisher Scientific) according to the manufacturer’s instructions. HER2-
positive and -negative cell lines were incubated with 5 mg/mL pHrodo
Deep Red-antibodies for 1 hour at 37
C and acquired by flow
cytometry on a CytoFLEX S flow cytometer (Beckman Coulter). An
increase in MFI indicates the presence of aHER2 antibodies in late
endosomal and lysosomal compartments. The MFI ratio was deter-
mined by dividing the MFI of pHrodo-incubated cells by the MFI of
unstained cells.
AACRJournals.org
Efficacious Exatecan-based DAR8 Antibody–Drug Conjugates
Bystander killing
For the co-culture–based bystander experiment, 10.000 HER2-
positive SKBR-3 and 2.000 HER2-negative MDA-MB-468 cells were
treated with trastuzumab-LP5 DAR8 or Enhertu at concentrations
ranging from 0.05 to 3 mg/mL. After 5 days, cells were stained
with LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific) and
HER2-PE (BioLegend) to distinguish between target-positive and
-negative cells. The percentage of viable HER2-positive and -neg-
ative cells was analyzed by flow cytometry on a CytoFLEX S flow
cytometer (Beckman Coulter).
DNA damage
The upregulation of DNA damage markers as response to TOP1-
inhibiting ADCs were analyzed by a flow cytometry–based readout.
For this, 30,000 HER2-positive SKBR-3 cells were incubated for
24 hours to 72 hours with 0.5 or 5 mg/mL of ADCs. After the end
of the incubation time, cells were stained with LIVE/DEAD Fixable
Aqua (Thermo Fisher Scientific) followed by fixation and permeabi-
lization using the BD Cytofix/Cytoperm Kit (BD Biosciences) accord-
ing to the manufacturer’s instructions. Intracellular staining of DNA
damage markers was performed using anti-cleaved PARP (Asp214)
PE, anti-H2AX (pSer139) AF647, and anti-active caspase 3 FITC (all
BD Biosciences) or respective isotype controls. Cells were acquired on
a CytoFLEX S flow cytometer (Beckman Coulter) and the percentage
of positive cells was determined.
Cell-cycle analysis
To investigate changes in cell cycle upon TOP1 inhibitor treatment,
30,000 HER2-positive SKBR-3 cells were incubated for 24 to 48 hour
with 20 nmol/L of ADCs (3 mg/mL) or small molecules if not otherwise
indicated. After the end of the incubation time, cells were permeabi-
lized in 70% ethanol on ice, followed by staining in FxCycle PI/RNAse
Staining Solution (Thermo Fisher Scientific). Cells were acquired on a
CytoFLEX S flow cytometer and PI fluorescence was analyzed on a
linear scale (Beckman Coulter).
Release of DAMPs as a surrogate marker for ICD in vitro
As an indirect measure for ICD, calreticulin-positive cells, HMGB1
and ATP release were quantified. For all readouts, 30,000 HER2-
positive cells were incubated with 20 nmol/L of exatecan, DXd or
derived ADCs (3 mg/mL) for 24 to 48 hours if not otherwise indicated.
Calreticulin staining of cells was performed using PE anti-calreticulin
antibody (Abcam) or respective isotype control. Lumit HMGB1
Human/Mouse Immunoassay (Promega) and RealTime-Glo Extra-
cellular ATP Assay (Promega) were used to quantify secreted HMGB1
and ATP into the media according to the manufacturer’s instructions.
Measurement of luminescence was performed on a microplate reader
Infinite 200 Pro (Tecan Group Ltd.).
In vivo efficacy experiments
The experiments were conducted in accordance with German
animal welfare law and approved by local authorities. A total of
2106 NCI-N87 cells were subcutaneously injected into the flank of
female CB17-Scid mice. Treatment was initiated when tumors reached
a mean tumor volume of 0.1–0.15 cm3. 10 animals per group were
treated once with either 0.25, 0.5, 1 or 2 mg/kg, trastuzumab-LP5
DAR8 or Enhertu. 5 animals per group were treated with vehicle or
with 2 mg/kg palivizumab-LP5 as Isotype control, as intravenous
injection after randomization into treatment and control groups.
Tumor volumes, body weights, and general health conditions were
recorded throughout the whole study.
Mol Cancer Ther; 2023 OF3
 Schmitt et al.
Data availability
The data generated in this study are available upon request from the
corresponding author.
Results
Design and evaluation of TOP1-based linker–payloads for highly
loaded and efficacious ADCs
We started our investigation with the selection of an appropriate
camptothecin derivative. For this, we tested the unconjugated pay-
loads, highlighted in orange in the linker–payload structures depicted
in Fig. 1, for their antiproliferative potency on a range of different cell
lines (Fig. 2; Supplementary Table S1).The figure clearly shows that of
the three TOP1 inhibitors tested in this study, exatecan is the most
potent with a factor of 2–10-fold potency difference in the IC50 value of
cell viability compared with SN38 and DXd. Next, a series of different
linker–payloads, shown in Fig. 3 based on exatecan, including a
maleimide-control ADC, has been synthesized. The dipeptidyl-
based linker–exatecan constructs LP1–5 carry the well-known VC
and valine–alanine (VA) peptides in combination with the self-
Figure 2.
Dose–response curves for DXd, SN38, and exatecan on a range of different cell lines.
OF4 Mol Cancer Ther; 2023
immolative PAB moiety that facilitates traceless release of exate-
can (38). The linker–payload LP6 based on maleimide chemistry as
the most commonly used conjugation strategy was generated as a
control. To assess whether the exatecan moiety must be tracelessly and
efficiently cleaved to unleash its full activity, we also synthesized
construct LP7 without the PAB moiety and the non-cleavable LP8.
The absence of the lipophilic PAB moiety in LP7 and LP8 has the
advantage of reduced linker–payload hydrophobicity to potentially
reduce ADC aggregation.
With LP1–8 in hands, we continued our studies with conjugation
of the linker–payload to trastuzumab, a HER2-targeting antibody
of the same amino acid sequence that is used in the antibody of
Enhertu (39). Antibody conjugation has been conducted in accordance
to previously published procedures (31). A large excess of the linker–
payloads and TCEP reduction agents has been used in those first
conjugation experiments to force the conjugation reaction and ensure
the highest possible conjugation yield. After conjugation and purifi-
cation from residual linker–payload, TCEP, and HMWS via semi-
preparative SEC into PBS, the corresponding ADCs were analyzed for
yield based on protein concentration before and after the conjugation,
MOLECULAR CANCER THERAPEUTICS
 Efficacious Exatecan-based DAR8 Antibody–Drug Conjugates

Figure 3.
Depiction of the linker–payload (LP) structures used herein. Functional elements are highlighted. Hydrophilic substituent, purple; conjugation handle, gray; cleavage
side, green; release handle, orange; exatecan, black.

DAR via mass spectrometry (MS), formation of undesired HMWS via
SEC and targeted antiproliferative effect on two different HER2-
positive (SKBR-3 and HCC-78) and one HER2-negative cell line
(Table 1; Supplementary Fig. S1).
Interestingly, after conjugation of the phosphonamidate-O-ethyl–
substituent linker–payload LP1 to trastuzumab, we observed only a
minor conversion of the antibody to a DAR of 0.28, even with a large
excess of 30 equivalents of LP1 with respect to the antibody. In addition
to this, we observed a high percentage of HMWS formation of almost
10% during the conjugation process (Table 1). To exclude slower
reaction kinetics of thiol addition to ethynylphosphonamidates,
compared with very fast maleimide conjugation (30, 40), being the
reason for this low conjugation yield, we also tried the maleimide-
based linker–payload LP6 for conjugation. However, also LP6 only
resulted in a very minor conjugation to a DAR of 0.12 in the
monomer fraction after conjugation with almost 20% of HMWS
being formed (Table 1).
Table 1 shows that incorporating PEG chains into the VC–PAB–
exatecan-based linker–payload systems can improve the conjugation
efficiency and simultaneously reduce aggregation of the derived ADCs.
Both effects are depending on the PEG length, as shown by linker–
payloads LP2, LP3, and LP5, with 2, 12, and 24 PEG monomer units,
respectively. It could be demonstrated that an increase in PEG length
led to a higher DAR, less aggregation during the conjugation process
and consequently to an increase in the yield of monomeric protein after
conjugation and to an increase in in vitro efficacy on the targeted cell
lines (Table 1; Supplementary Fig. S1). Switching from a VC LP3 to a
VA LP4 did not improve the conjugation behavior (Table 1). Only the
PEG24-carrying linker–payload LP5 led to full conjugation of tras-
tuzumab to a DAR8 ADC without any sign of HMWS being formed
during the conjugation process resulting in the highest in vitro cyto-
toxicity on HER2-positive SKBR-3 and HCC-78 among all tested
linker–payloads. We also synthesized ADCs from linker–payload LP7,
which is lacking the PAB moiety. Interestingly, LP7 yields ADCs with a

Table 1. Overview of the properties of the ADCs, synthesized from the HER2-targeting trastuzumab conjugated to LP1–8.

Yield after DAR (MS) Cell killing SKBR-3 Cell killing HCC-78
purification After
based on purification Max. Max.
protein from HMWS after EC50 amount of EC50 amount of
LP Linker–payload concentration HMWS conjugation (%) (ng/mL) dead cells (ng/mL) dead cells

LP1 P5(OEt)–VC–PAB–exatecan 59% 0.28 9.9% 251 37% >3,000 N/A

LP2 P5(PEG2)–VC–PAB–exatecan 82% 5.51 2.3% n.d. n.d. n.d. n.d.
LP3 P5(PEG12)–VC–PAB–exatecan 80% 6.36 3.9% 15.5 86% 144.1 91%
LP4 P5(PEG12)–VA–PAB–exatecan 84% 4.39 7.5% 18.0 78% 121.2 68%
LP5 P5(PEG24)–VC–PAB–exatecan 99% 8.00 0.9% 12.5 96% 97.6 88%
LP6 MC–VC–PAB–exatecana 75% 0.12 19.2% 265 14% >3,000 N/A
LP7 P5(PEG12)–VA–exatecan 77% 7.89 1.0% 114.7 44% >3,000 N/A
LP8 P5(PEG12)–exatecan 97% 7.19 4.0% 182.4 35% >3,000 N/A

Note: The conjugation has been performed with excess linker–payload (30 eq. with respect to the antibody) and TCEP (10 eq. with respect to the antibody). After
purification, the ADCs have been analyzed for yield based on protein concentration before and after the conjugation, DAR via MS, ADC aggregation via analytic
SEC and antiproliferative effect on HER2-positive cell-lines (SKBR-3 and HCC-78). The antiproliferative potency of the ADCs on the HER2-positive cells is reported
as an EC50 value of cell viability in ng/mL and the total amount of affected cells in percentage. None of the ADCs showed any effect on the HER2-negative cell line
(MDA-MB-468, Supplementary Fig. S1).
a
Conjugation conditions for LP6 were slightly adjusted to more typical maleimide reaction conditions: Reaction was conducted in PBS at pH 7.4 at room temperature
for 2 hours; n.d., not determined; N/A, not applicable.

AACRJournals.org Mol Cancer Ther; 2023 OF5

 Schmitt et al.

very high DAR and almost no HMWS being formed during conju-
gation, in contrast with LP4, which is the same linker–payload
structure but includes the PAB moiety. However, we observed that
ADCs from LP7 and LP8, even though high in DAR, showed only
modest anticancer activity in the antigen-positive cell lines (Table 1;
Supplementary Fig. S1).
Biophysical and in vivo characterization of the two best
performing exatecan-based linker–payloads
Next, we continued our efforts in designing novel camptothecin-
based linker–payload structures with a more thorough in vitro and
in vivo investigation of ADCs originating from linker–payload LP3
and LP5, carrying either 12 or 24 PEG units, respectively. To allow for a
fair comparison between the linker systems, DAR8 ADCs were
synthesized out of both linker–payload structures (Fig. 4A). For this,
equivalents of LP3 were increased from 30, that were used to produce
the results in Table 1, to 50 and the monomeric ADCs were isolated via
preparative SEC. The desired ADCs were characterized via SEC and
hydrophobic interaction chromatography (HIC; Fig. 4B). Trastuzu-
mab-LP5 DAR8 demonstrated best characteristics, because it had the
highest degree of monomer content compared with trastuzumab-LP3
and Enhertu, judged by SEC. Interestingly, the overall hydrophobicity
increase of the DAR8 constructs compared with the unconjugated
antibody was negligible, judged by the HIC retention time (Supple-
mentary Fig. S2) and similar HIC retention times were measured for
trastuzumab-LP3 DAR8, trastuzumab-LP5, and Enhertu (Fig. 4B).
The aggregation behavior of trastuzumab-based DAR8 ADCs from
LP3 and LP5 was more thoroughly monitored over several weeks at
low (4
C) and physiological temperatures (37
C) and Enhertu was
added as a comparison (Fig. 4C; Supplementary Fig. S2). The results
clearly show that ADCs conjugated to LP3 have a stronger tendency to
aggregate under both conditions compared with Enhertu and ADCs
conjugated to LP5. It should be noted that DAR8 ADCs from LP5
showed the least tendency to form HMWS under the tested conditions,
even compared with Enhertu.
To compare ADCs from LP3 and LP5 in their in vivo PK behavior,
Sprague Dawley rats were treated with ADCs carrying 8 linker–
payload molecules, and their clearance behavior was monitored over
time via analysis of blood samples. It must be noted that both
experiments have been performed with different antibodies conjugat-
ed to LP3 and LP5. However, because both antibodies are not cross-
reactive in rodents, it is anticipated that the influence on the clearance
behavior of the ADCs can be neglected. The samples at various time
points were analyzed by ELISA for total ADC with an antibody capture
step and for intact ADC, capturing with an anti-exatecan antibody.
The high overlap between those two curves again highlights the great
serum stability of the ehynylphosphonamidate linker structure and is
in line with previous reports (31, 32). Interestingly, the stronger
aggregation tendency of ADCs synthesized from LP3 did not drasti-
cally increase the in vivo clearance of the ADCs (Fig. 4D). Moreover,
HIC analysis of ADCs from LP3 showed only a modest increase in
overall hydrophobicity compared with MMAE-based ADCs (Supple-
mentary Fig. S3).
In vitro head-to-head comparison of the best performing linker–
payload to enhertu
With our main goal being to develop a novel superior camptothecin-
based linker–payload structure, we continued our investigations with
ADCs that originate from LP5, because this linker–payload structure
combines high conjugation yields in DAR and antibody recovery, very
low aggregation tendency, highest in vitro activity and excellent in vivo
stability and clearance behavior. To ensure manufacturability also on a
higher scale, we first investigated how many equivalents of LP5 are
needed to synthesize DAR8 ADCs. On the basis of previous experience,
we determined the DAR as a function of equivalents of LP5 and
equivalents of TCEP in the conjugation reactions in a pH 8.3 buffer

Figure 4.
Head-to-head comparison between DAR8 ADCs from LP3 and LP5 and Enhertu. A, Structure of DAR8 ADCs from LP3 and LP5. B, Comparison of the physicochemical
properties of trastuzumab-LP5 (orange), trastuzumab-LP3 (purple), and Enhertu (gray). SEC analysis for the formation of aggregation (top) and HIC analysis
to investigate overall hydrophilicity of the DAR8 constructs (bottom). C, Monitoring of increase in aggregation over 4 weeks of trastuzumab-LP3 (orange),
trastuzumab-LP5 (purple), and Enhertu (gray). All three ADCs were formulated in the same buffer system at 1 mg/mL. Samples were analyzed by SEC for HMWS after
1, 2, and 4 weeks of incubation at 4
C (left) and 37
C, right. Raw SEC chromatograms are shown in Supplementary Fig. S3. Aggregates were normalized to day 0 by
subtraction, because trastuzumab-LP5, already contained 8% HMWS at day 0. D, PK analysis of Sprague Dawley rats that were treated at day 0 with brentuximab-LP5
(left, purple) or IgG1-LP5 (right, orange) at day 0. Samples were drawn after several time points throughout the 3 week study period (9 animals per group, 3 animals
were sampled per time point). Shown are mean and SD out of the three animals. The serum samples were analyzed by ELISA for total mAb (dotted line) and intact
ADC (straight line). Black dotted line corresponds to the lower limit of quantification of the assays.

OF6 Mol Cancer Ther; 2023 MOLECULAR CANCER THERAPEUTICS


system at 10 mg/mL (31). We found out that under those conditions, as
less as 10 equivalents with respect to the mAb (1.25 eq. per Cys) are
enough to generate a DAR8 ADC (Supplementary Fig. S4). Moreover,
we tried to reduce the pH of the conjugation reaction. Here, it could be
shown that the reaction kinetics are pH-dependent. Full conversion to
DAR8 is still possible at pH 7.8, when the reaction time is increased to
24 hours (Supplementary Fig. S4).
After optimization of the conjugation conditions with LP5, the
conjugation to trastuzumab was scaled to 10 grams of mAb and the
DAR8 ADC was purified via tangential flow filtration only. Analytic
data of the upscaling runs were identical to what has been observed on
small scale. The DAR8 trastuzumab-LP5 ADC has been further
investigated in head-to-head comparisons with Enhertu. For this
purpose, Enhertu has been purchased in pharmaceutical grade. First,
a broader panel of HER2-positive cell lines and one HER2-negative cell
line were treated with either trastuzumab-LP5 or Enhertu. The results
show that the in vitro efficacy is equal or superior in all HER2-positive
cell lines (Fig. 5A). Less unspecific killing was observed in the non-
targeted antigen-negative cell line L-540. In general, we were able to
observe an expected correlation between ABCs and in vitro cytotox-
icity of both ADCs (Fig. 5B). Of note, only minor differences in
binding on SKBR-3 (KD trastuzumab-LP5: 475.1 ng/mL, Enhertu:
420.6 ng/mL) and internalization on HER2-positive and -negative cell
lines were observed between trastuzumab-LP5 and Enhertu (Fig. 5C).
Next, we investigated the bystander capacity, a potential feature of
ADCs to compensate for heterogenous target expression, in depen-
dency of the concentration of trastuzumab-LP5 DAR8 and Enhertu.
Thereby, non-targeted HER2-negative MDA-MB-468 cells (47 ABCs)
were co-cultured with HER2-positive SKBR-3 (790520 ABCs) fol-
lowed by the assessment of cell viability of the HER2-negative cell only
via flow cytometry. The MDA-MB-468 cells were only affected in
viability when co-cultured with SKBR-3. A tendency toward lower IC50
values and higher number of cells being affected was visible for
trastuzumab-LP5 compared with Enhertu (Fig. 5D). Moreover, we
characterized the mechanism of cell killing induced by trastuzumab-
LP5 and Enhertu in more detail. We have shown by flow cytometry
that trastuzumab-LP5 DAR8 and Enhertu are both inducing DNA
damage resulting from TOP1 inhibition to a similar extent after
treatment of SKBR-3 cells with 0.5 mg/mL of the ADCs as shown by
the upregulation of phosphorylated histon 2AX, activated caspase 3,
and cleaved PARP (Fig. 5E; Supplementary Fig. S6). Another mech-
anism of action of TOP1 inhibition is the induction of cell-cycle arrest,
which has been shown in cells treated with trastuzumab-LP5 DAR8,
Enhertu and also free payloads (Fig. 5F; Supplementary Fig. S6). Cells
were arrested mainly in S phase, but also in G2–M phase at selected
concentrations and incubation times. Another important feature for
cytotoxic payloads (also described for TOP1 inhibitors) is the
induction of ICD, a process by which the drug-induced apoptosis
of a tumor cell releases DAMPs and thereby stimulates the immune
system. For this purpose, SKBR-3 cells were treated with Enhertu,
trastuzumab-LP5 or the respective payloads DXd and exatecan,
followed by the quantification of cells exposing calreticulin on the
surface and releasing ATP and HMGB1 (Fig. 5G; Supplementary
Fig. S7), all of which are DAMPs and surrogate markers for ICD.
Indication for induction of ICD was shown by an increase in
exposed calreticulin on the surface and ATP and HMGB1 release
for both ADCs in a similar manner.
Stability and in vivo PK head-to-head comparison with enhertu
The high stability of ethynylphosphonamidate-linked ADCs, in
particular in comparison with maleimide chemistry, has been
AACRJournals.org
Efficacious Exatecan-based DAR8 Antibody–Drug Conjugates
described earlier (30–32) and could be confirmed here in the direct
comparison of retained DAR after several days of incubation in rat
serum (Fig. 6A). Moreover, we performed an in vivo stability head-to-
head comparison between trastuzumab-LP5 DAR8 and Enhertu.
Here, mice were treated with 20 mg/kg of each ADC at day 0 and
blood samples were drawn after 1, 3, 5, and 7 days after treatment.
Serum samples were analyzed by ELISA for total ADC exposure,
showing a similar in vivo clearance behavior of trastuzumab-LP5
compared with Enhertu, with a slightly higher exposure for tras-
tuzumab-LP5, 7 days after treatment. Moreover, the ADCs were
captured from the sera by immunoprecipitation and analyzed via
MS for DAR. The results clearly indicate that the ex vivo stability
also translates to an in vivo setting, showing that trastuzumab-LP5
is still connected to 8 linker–payload molecules after one week of
circulation, whereas Enhertu’s DAR is reduced to approximately 5
(Fig. 6B). The main route of drug loss that has been observed for
Enhertu is via retro-Michael addition, leading to the detection of the
unmodified cysteines by MS.
Encouraged by these results, we wanted to investigate the influence
of the reduction in DAR on the in vitro efficacy of the constructs,
because we have already shown in Table 1 that a lower DAR can
lead to a reduction in cell killing efficiency. Therefore, we took the
serum-stressed samples containing trastuzumab-LP5 DAR8 and
Enhertu and performed a cell viability experiment on a HER2-positive
and a HER2-negative cell line (Fig. 6C). The results show that the
reduction in DAR that originates from a retro-Michael–based insta-
bility of Enhertu also directly leads to a reduction in cell killing of the
antigen-positive cell line after prolonged serum incubation. As
expected, this effect is much less pronounced for the stably conjugated
trastuzumab-LP5 DAR8.
In vivo efficacy head-to-head comparison with enhertu and PK
analysis
Next, we performed an in vivo experiment, in which we compared
the efficacy of trastuzumab-LP5 DAR8 to that of Enhertu over 4
different dose levels. 10 mice per group were implanted with tumors
derived from the HER2-positive N87-cell line and treated once with
either 0.25, 0.5, 1 or 2 mg/kg at day 0, once the tumors have reached a
volume of 0.1–0.15 cm3. The N87-cell line was chosen because no
significant difference in in vitro efficacy between both ADCs was
observed (Fig. 5A). We anticipated that thereby the true impact of the
linker technology can be assessed without being biased by the more
potent payload that was chosen in LP5 compared with deruxtecan. The
results show a clear benefit in efficacy for trastuzumab-LP5 over all 4
dose levels tested (Fig. 6D). It should be highlighted that trastuzumab-
LP5 DAR8 is more effective at 0.5 mg/kg than Enhertu at 1 mg/kg.
Moreover, at 1 mg/kg, no complete remissions were observed in
Enhertu in contrast with 80% for trastuzumab-LP5 DAR8. At the
highest dose level of 2 mg/kg, a non-targeted isotype control has been
included, which did not show an effect with similar tumor growths
than in the vehicle control, clearly underlining the target selectivity of
LP5 when conjugated to an antibody.
Finally, we conducted a more comprehensive PK analysis of ADCs
based on LP5 in Sprague Dawley rats. Previously, high DAR ADCs of
payloads such as MMAE have been shown to rapidly clear from
circulation (41). Here, two groups of rats were treated with 10 mg/kg
of either an unconjugated IgG1 or a DAR8 conjugate of that antibody
(IgG1-LP5 DAR8). Blood samples from those groups were taken over a
time course of three weeks and analyzed via ELISA for total antibody
and total and intact ADC. Remarkably, we observed a very similar
clearance profile of the IgG1 antibody, and the same antibody
Mol Cancer Ther; 2023 OF7
 Schmitt et al.

Figure 5.
Cell-based in vitro efficacy experiments of trastuzumab-LP5 DAR8 (orange) or isotype-LP5 DAR8 (black) compared with Enhertu (gray). A, Cell viability on seven
HER2-positive cell lines (N87, SKBR-3, HCC-78, SK-OV-3, OE-19, SK-GT-2, and HCC-1569) and one HER2-negative cell line L-540. B, Correlation between HER2-
receptor level on the tested cell lines and antiproliferative effect (EC50 in ng/mL) for trastuzumab-LP5 DAR8 (left, orange) and Enhertu (gray, right). C, Binding of
trastuzumab-LP5 and Enhertu on SKBR-3 and internalization on SKBR-3, N87 (HER2þ) and MDA-MB-468 (HER2). D, Bystander killing of non-targeted HER2-
negative MDA-MB-468 cells in co-culture with HER2-positive SKBR-3 (right). Shown is cell viability in dependency of the ADC concentrations. No effect was observed
in MDA-MB-468 only cultures (left). EC) Relative quantification of histone H2AX phosphorylation (left), active caspase 3 (middle) and cleaved PARP (right) after
treatment of SKBR-3-cells with 500 ng/mL of the ADCs for 3 days versus untreated. D–F, Relative quantification of cells in S-, sub-G0, G0–G1, and G2–M phase
after treatment with 3,000 ng/mL of the ADCs for 2 days versus untreated. E–G, Secretion of DAMPs as a surrogate marker for measurement of immunogenic
cell death as indicated by calrecticulin exposure on cell surface (left), ATP release (middle), and HMGB1 release (right). Shown are results after treatment
with 3,000 ng/mL (¼ 20 nmol/L) of the trastuzumab-LP5 DAR8 (orange, solid) or Enhertu (gray, solid) and with 20 nmol/L of unconjugated exatecan (orange,
checkered) or DXd (gray, checkered) for 2 days versus untreated (black). Graphs show means of n ¼ 2  SEM.

OF8 Mol Cancer Ther; 2023 MOLECULAR CANCER THERAPEUTICS

 Efficacious Exatecan-based DAR8 Antibody–Drug Conjugates

Figure 6.
Stability experiments, in vivo efficacy and PK analysis of trastuzumab-LP5 DAR8, (orange) compared with isotype-LP5 DAR8 (yellow), vehicle (black), and
Enhertu (gray). A, DAR ratio measured by MS after 0, 1, 3, and 7 days of incubation in at least 80% rat serum at 37
C. B, PK analysis of blood samples from mice
being treated with 20 mg/kg. Samples have been drawn after 1, 3, 5, and 7 days of circulation. Exposure by total ADC ELISA (top). DAR measured by MS
(bottom). C, Cell viability experiments of ADC samples after 0, 1, 3, and 7 days of incubation in at least 80% rat serum at 37
C on the HER2-positive cell line
SKBR-3 (left) or HER2-negative cell line MDA-MB-468 (right). D, Female SCID mice have been implanted with cell-culture–derived xenograft model based on
N87 cells in Matrigel. Treatment was started once when tumor volumes reached 0.1–0.15 cm3. Mice were treated at day 0 with a single dose of either vehicle, 2,
1, 0.5, and 0.25 mg/kg trastuzumab-LP5 DAR8, or Enhertu. At the highest dose of 2 mg/kg, a non-targeting isotype control ADC Isotype-LP5 DAR8 was
included. Each group consisted of n ¼ 10 females each. F, Sprague Dawley rats were treated at day 0 with 10 mg/kg of either IgG1 (left) or IgG1-LP5 DAR8
(right) at day 0. Samples were drawn after several time points throughout the 3 week study period (9 animals per group, 3 animals were sampled per time
point). Shown are mean and SD out of the three animals. The serum samples were analyzed by ELISA for total mAb/total ADC (dotted line) and intact ADC
(straight line). G, DAR measured by MS of blood samples from rats being treated with 10 mg/kg of IgG1-LP5 DAR8. Black dotted line corresponds to the lower
limit of quantification of the assays.

conjugated to 8 molecules of LP5, judged by the similarity of the

profiles for total ADC and total antibody and of the AUC values
(Fig. 6E). This again highlights the negligible influence of the
conjugation of 8 molecules of linker–payload LP5 to the antibody
leading to a high DAR ADC with mAb-like properties. Once more,
excellent stability could be confirmed by overlapping total ADC and
intact ADC. This could be verified by an MS assay, which revealed a
DAR8 circulating in rats even 21 days after administration of the
ADC (Fig. 6F).
Discussion
With this study, we aimed to develop a superior TOP1 Inhib-
itor-based linker–payload platform for the construction of high
DAR ADCs. Our studies demonstrate antibody-like properties
for highly loaded phosphonamidate-linked exatecan-containing
ADCs, thus providing a promising alternative for existing link-
er–payload structures used in marketed ADCs such as Trodelvy
and Enhertu.

AACRJournals.org Mol Cancer Ther; 2023 OF9

 Schmitt et al.
On the basis of our findings of exatecan being more potent than
SN38 and DXd and with the intention to design TOP1 inhibitor-based
ADCs with superior efficacy, we decided to construct linker–payload
systems that use exatecan as the cytotoxic payload. With the aim to
compensate for a potential toxicity risk, which is often associated with
ADCs based on more potent payloads and linker instability (13, 14, 42),
we decided to make use of ethynylphosphonamidates for antibody
conjugation. This linker class has been previously shown to exhibit
superior stability in head-to-head comparisons with approved
ADCs (32, 43, 44). Hence, we synthesized several linker–exatecan
constructs with and without the traceless PAB-release unit in the
presence or absence of different dipeptidyl cleavage sides, with the
addition of PEG solubilizing groups of various lengths. VC–PAB–
exatecan constructs with shorter PEG chains than 12 units showed a
strong aggregation tendency and led to low conjugation yields. This
stands in strong contrast with what has previously been observed with
other ethynylphosphonamidate-based linker–payloads (30, 32, 40)
and led to our conclusion that the VC–PAB–exatecan moiety, as a
very hydrophobic linker–payload group itself, exhibits the following
challenges: (i) Low aqueous solubility leading to decreased available
linker–payload concentrations under the aqueous conjugation con-
ditions and (ii) ADCs, once formed, tend to form HMWS, leading
again to reduced DARs in the monomeric fraction. Attempts to reduce
this aggregation by switching from a VC LP3 to a VA linker–payload
LP4 did not improve the conjugation behavior, which is in agreement
with previous reports on the differences between those two dipep-
tides (41). To reduce the overall hydrophobicity of the linker–pay-
loads, we incorporated PEG chains at the phosphonamidates-O-
substituent (31). This strategy has been previously reported for ADCs
to improve the PK profile of MMAE-based ADCs, because it reduces
clearance from circulation mediated by the overall hydrophobicity of
the ADC (18, 19). Notably, selective potency on the two tested HER2-
positive, targeted cancer cell lines also increased with longer PEG
chains, observable by a shift of the IC50 value to lower concentration
and an increase in the maximum amount of cells that have been
affected by the ADC. Most likely this is a function of the higher drug-
load that could be achieved with higher PEG length and is in line with
previous reports on higher DAR-ADCs having a higher potency
in vitro (41, 45).
ADC aggregation arising during the conjugation process was a
major issue that prevented the formation of high DAR ADCs for some
of the constructs. It should be noted that HMWS formation was not
observed in the past when VC–PAB–MMAE or VC–PAB–MMAF was
used instead of VC–PAB–exatecan, even with shorter PEG substitu-
ents at the ethynylphosphonamidate (30, 32). The observation on
aggregation being induced by the VC–PAB–exatecan moiety is sup-
ported by studies of Burke and colleagues (20), who have also reported
strong tendencies for HMWS formation with other maleimide–VC–
PAB–camptothecin constructs. For deruxtecan, the linker–payload of
Enhertu with a very similar camptothecin core structure compared
with exatecan but different in linker, there have not been reports on
problematic aggregation behavior even at high DARs (22). We there-
fore suspected that the origin of the aggregation and solubility issue
lays in the combination of exatecan with the p-aminobenzyl moiety,
leading to moieties that combine several p-electron systems, poten-
tially leading to flat, ridged PAB–exatecan moieties. Strong p–p
interactions between molecules that carry several aromatic cores are
generally known to impair solubility (46). Indeed, LP7, which is in
comparison with LP4 lacking the PAB moiety is not inducing aggre-
gation even at high DAR. However, ADCs from LP7 are also clearly
OF10 Mol Cancer Ther; 2023
reduced in in vitro efficacy in comparison with conjugates that carry
the PAB release handle. This observation is in line with early inves-
tigations of Trail and coworkers that the PAB moiety can be crucial for
efficient release of the payload (33). Very similar behavior was
observed for the non-cleavable linker–payload LP8, clearly indicating
the necessity of an efficient release handle for a potent antibody-
mediated delivery of exatecan into the targeted cell.
In a head-to-head comparison of the suboptimal PEG12-based LP3
with the superior PEG24-based LP5, we have furthermore shown that
conjugation of 8 VC–PAB–exatecan molecules to an antibody is
neither drastically increasing the overall hydrophobicity of the anti-
bodies, judged by HIC retention, nor the in vivo clearance rates, which
makes aggregation during conjugation and low conjugation yields the
main challenge to be solved for VC–PAB–exatecan. This is contrary to
the observation by us and others that VC–PAB–MMAE DAR8 ADCs
clear rapidly from circulation in vivo, when not adequately balanced
with solubility enhancers (18, 41). We interpret this as being another
hint toward our hypothesis that not the overall hydrophobicity of VC–
PAB–exatecan-based ADCs lead to aggregation and low conjugation
but rather the strong tendency of larger aromatic molecules to build
strong intermolecular p–p interactions. In previous work, we have
mainly used the P5(PEG) moieties to improve in vivo PK of high-DAR
ADCs (31, 32). Here, we demonstrate that such P5(PEG) building
blocks can also be applied to efficiently reduce ADC aggregation and
facilitate conjugation, which generally underlines the beneficial fea-
tures of branched PEG-based linker systems.
With the identification of phosphonamidate-linked P5(PEG24)–
VC–PAB–exatecan LP5 as an optimal linker–payload system, we
improved the conjugation conditions to be able to use as little as
1.25 equivalents per cysteine to generate a DAR8 ADC from an IgG1
antibody. Next, a comprehensive in vitro head-to-head comparison
between trastuzumab-LP5 DAR8 and Enhertu has been performed.
We could confirm that both ADCs exhibit the same mechanism
of cell killing as a result of TOP1 inhibition, demonstrated by upre-
gulation of DNA damage markers such as phosphorylated histon 2AX,
activated caspase 3, and cleaved PARP (47). Moreover, in accordance
with literature data, we showed both S and G2–M arrest as response
to TOP1 inhibition (48, 49). We also investigated the capability of
TOP1-based ADCs to elicit ICD, a process by which a cytotoxic drug
induces apoptosis of a tumor cell that causes stimulation of the
immune system. Increase of immunostimulatory DAMPs has been
previously described to accompany and precede ICD and serves as a
surrogate marker for the latter (50, 51). Immunostimulatory activity
has been described for Enhertu and could be confirmed in the
current study via an in vitro experiment. Such activation of the
immune system can lead to a long-lasting protective antitumor
immunity and opens a highly promising road for combination of
ADCs with immuno-oncology agents (52, 53). The release of
DAMPs from trastuzumab-LP5 in similar quantities than Enhertu
indicates the immunostimulatory effect of exatecan when delivered
via LP5 conjugated to a targeted antibody.
Higher in vitro potency of trastuzumab-LP5 compared with
Enhertu could be observed in some cell lines. Because we demon-
strated similar HER2 binding for both ADCs, we attribute this to
differences in potency of the attached payloads, but not to an
influence of the attached linker on the properties of the antibody.
Bystander killing is considered to play a key role in the effective
treatment of solid tumors to compensate for heterogenous target
expression (54). Enhertu has been described to exhibit highly potent
bystander activity, in particular when compared with ADCs with
MOLECULAR CANCER THERAPEUTICS

other camptothecin-based linker–payload systems and to trastuzu-
mab-DM1 (23). Here, we even observed a slightly higher bystander
killing effect for trastuzumab-LP5 over Enhertu. Most importantly,
we were able to show a high increase of trastuzumab-LP5 in serum
stability of the linker in vitro and in vivo. This results in retained
in vitro activity of trastuzumab-LP5 DAR8 even after several days of
serum stress, whereas cell killing was drastically reduced for
Enhertu in this setting. The effect of DAR reduction and unstable
linker technology in circulation on the safety of an ADC is still
under debate (1). However, those results rather suggest a retained
efficacy over time in circulation of ADCs conjugated to LP5, in
particular when compared with unstably conjugated maleimides as
used in Enhertu.
Moreover, we were able to show that the superiority of trastu-
zumab-LP5 in cell killing, bystander activity, and serum stability of
the linker in vitro and in vivo also translates into improved in vivo
efficacy. Trastuzumab-LP5 DAR8 revealed a more potent targeted
effect over four tested dose levels, with a drastically increased
activity even when half of the dose has been administered. We
explain this with a slightly better PK profile, with less clearance and
very importantly higher in vivo stability, which taken together helps
to deliver the payload more efficiently to the site of the tumor. We
conclude that those features can be attributed to the linker prop-
erties rather than exatecan being more potent payload compared
with DXd, because we chose a cell line in which both ADCs had a
highly comparable efficacy in vitro.
Finally, it could be shown in an in vivo PK study that LP5 can be used
to synthesize DAR8 ADCs with antibody-like clearance behavior in
combination with highest stability, judged by the detection of a fully
conjugated DAR8 ADC after 21 days of circulation in a living
organism. We believe that phosphonamidate-linked exatecan pay-
loads LP5 can be used to reduce last drawbacks of the already well-
functioning deruxtecan platform used in Enhertu for a next-
generation of TOP1-based ADCs of highest therapeutic activity over
a broad span of different tumor antigen targets.
Authors’ Disclosures
S. Schmitt reports grants from Federal Ministry for Economic Affairs and Energy
(EXIST Forschungstransfer II), Federal Ministry for Economic Affairs and Energy
(German Accelerator), Federal Ministry of Education and Research (Rahmenpro-
gramm Gesundheitsforschung), and LMU Munich outside the submitted work; as
well as reports a patent for WO2023083919 pending; and Tubulis GmbH sponsored
the work and is owner of the relevant patents listed above; and reports employment
with Tubulis GmbH. P. Machui reports grants from Federal Ministry for Economic
Affairs and Energy (EXIST Forschungstransfer II), Federal Ministry for Economic
Affairs and Energy (German Accelerator), Federal Ministry of Education and
Research (Rahmenprogramm Gesundheitsforschung), and LMU Munich outside
the submitted work; as well as reports a patent for WO2023083919 pending; and
Tubulis GmbH sponsored the work and is owner of the relevant patents listed above;
and reports employment with Tubulis GmbH. I. Mai reports grants from Federal
Ministry for Economic Affairs and Energy (EXIST Forschungstransfer II), Federal
Ministry for Economic Affairs and Energy (German Accelerator), Federal Ministry of
Education and Research (Rahmenprogramm Gesundheitsforschung), and LMU
Munich outside the submitted work; as well as reports a patent for WO2023083919
pending; and Tubulis GmbH sponsored the work and is owner of the relevant patents
listed above; and reports employment with Tubulis GmbH. S. Herterich reports grants
from Federal Ministry for Economic Affairs and Energy (EXIST Forschungstransfer
II), Federal Ministry for Economic Affairs and Energy (German Accelerator), Federal
Ministry of Education and Research (Rahmenprogramm Gesundheitsforschung),
and LMU Munich outside the submitted work; and Tubulis GmbH sponsored the
work and is owner of the relevant patents listed above; as well as reports employment
with Tubulis GmbH. S. Wunder reports grants from Federal Ministry for Economic
Affairs and Energy (EXIST Forschungstransfer II), Federal Ministry for Economic
AACRJournals.org
Efficacious Exatecan-based DAR8 Antibody–Drug Conjugates
Affairs and Energy (German Accelerator), Federal Ministry of Education and
Research (Rahmenprogramm Gesundheitsforschung), and LMU Munich outside
the submitted work; and Tubulis GmbH sponsored the work and is owner of the
relevant patents listed above; as well as reports employment with Tubulis GmbH. P.
Cyprys reports grants from Federal Ministry for Economic Affairs and Energy (EXIST
Forschungstransfer II), Federal Ministry for Economic Affairs and Energy (German
Accelerator), Federal Ministry of Education and Research (Rahmenprogramm
Gesundheitsforschung), and LMU Munich outside the submitted work; and Tubulis
GmbH sponsored the work and is owner of the relevant patents listed above; as well as
reports employment with Tubulis GmbH. M. Gerlach reports grants from Federal
Ministry for Economic Affairs and Energy (EXIST Forschungstransfer II), Federal
Ministry for Economic Affairs and Energy (German Accelerator), Federal Ministry of
Education and Research (Rahmenprogramm Gesundheitsforschung), and LMU
Munich outside the submitted work; and Tubulis GmbH sponsored the work and
is owner of the relevant patents listed above; as well as reports employment
with Tubulis GmbH. P. Ochtrop reports grants from Federal Ministry for Economic
Affairs and Energy (EXIST Forschungstransfer II), Federal Ministry for Economic
Affairs and Energy (German Accelerator), Federal Ministry of Education and
Research (Rahmenprogramm Gesundheitsforschung), and LMU Munich outside
the submitted work; and Tubulis GmbH sponsored the work and is owner of the
relevant patents; as well as reports employment by Tubulis GmbH. C.P.R. Hack-
enberger reports grants from Federal Ministry of Education and Research (Rahmen-
programm Gesundheitsforschung), Leibniz Competition (SAW), and German Sci-
ence Foundation (DFG) outside the submitted work; as well as reports a patent for
WO2018041985 issued. D. Schumacher reports grants from Federal Ministry for
Economic Affairs and Energy (EXIST Forschungstransfer II), Federal Ministry for
Economic Affairs and Energy (German Accelerator), Federal Ministry of Education
and Research (Rahmenprogramm Gesundheitsforschung), and LMU Munich outside
the submitted work; as well as reports a patent for WO2018041985 issued and
WO2023083919 pending; and Tubulis GmbH sponsored the work and is owner of the
relevant patents listed above; and reports employment with Tubulis GmbH. J. Helma
reports grants from Federal Ministry for Economic Affairs and Energy (EXIST
Forschungstransfer II), Federal Ministry for Economic Affairs and Energy (German
Accelerator), Federal Ministry of Education and Research (Rahmenprogramm
Gesundheitsforschung), and LMU Munich outside the submitted work; as well as
reports a patent for WO2018041985 issued and WO2023083919 pending; and
Tubulis GmbH sponsored the work and is owner of the relevant patents listed above;
and reports employment with Tubulis GmbH. A.M. Vogl reports grants from Federal
Ministry for Economic Affairs and Energy (EXIST Forschungstransfer II), Federal
Ministry for Economic Affairs and Energy (German Accelerator), Federal Ministry of
Education and Research (Rahmenprogramm Gesundheitsforschung), and LMU
Munich outside the submitted work; as well as reports a patent for WO2023083919
pending; and Tubulis GmbH sponsored the work and is owner of the relevant patents
listed above; and reports employment with Tubulis GmbH. M.-A. Kasper reports
grants from Federal Ministry for Economic Affairs and Energy (EXIST Forschung-
stransfer II), Federal Ministry for Economic Affairs and Energy (German Acceler-
ator), Federal Ministry of Education and Research (Rahmenprogramm Gesundheits-
forschung), and LMU Munich outside the submitted work; as well as reports a patent
for WO2023083919 pending and WO2018041985 issued; and Tubulis GmbH
sponsored the work and is owner of the relevant patents listed above; and reports
employment with Tubulis GmbH.
Authors’ Contributions
S. Schmitt: Data curation, formal analysis, investigation, writing–review and
editing. P. Machui: Formal analysis, investigation. I. Mai: Data curation, investiga-
tion. S. Herterich: Data curation, investigation. S. Wunder: Investigation. P. Cyprys:
Investigation. M. Gerlach: Methodology. P. Ochtrop: Conceptualization.
C.P.R. Hackenberger: Conceptualization, methodology, writing–review and edit-
ing. D. Schumacher: Supervision, funding acquisition, methodology, project admin-
istration. J. Helma: Conceptualization, supervision, funding acquisition, methodol-
ogy. A.M. Vogl: Conceptualization, supervision, methodology, writing–review and
editing. M.-A. Kasper: Conceptualization, data curation, supervision, investigation,
visualization, methodology, writing-original draft.
Acknowledgments
The authors thank Danila Hauswald for excellent technical assistance in antibody
purification. This work was supported by grants from the German Federal Ministry
for Economic Affairs and Energy and the European Social Fund with grants (to
D. Schumacher and J. Helma; EXIST FT I); and the Bavarian Ministry of Economic
Mol Cancer Ther; 2023 OF11
 Schmitt et al.
Affairs, Regional Development and Energy with grants (to D. Schumacher, J. Helma,
and C.P.R. Hackenberger; m4-Award).
The publication costs of this article were defrayed in part by the payment of
publication fees. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
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