Cetuximab-Triptolide Conjugate Suppresses the Growth of EGFR-Overexpressing Lung Cancers through Targeting RNA Polymerase II

Full text of DOI:10.1016/j.omto.2020.07.001

Original Article
Cetuximab-Triptolide Conjugate Suppresses
the Growth of EGFR-Overexpressing Lung Cancers
through Targeting RNA Polymerase II
Keqiang Zhang,1,4 Yuelong Ma,2,4 Yuming Guo,3 Ting Sun,1 Jun Wu,3 Rajendra P. Pangeni,1 Min Lin,2 Wendong Li,1
David Horne,2 and Dan J. Raz1
1Division of Thoracic Surgery, City of Hope National Medical Center, Duarte, CA, USA; 2Department of Molecular Medicine, City of Hope National Medical Center,
Duarte, CA, USA; 3Division of Comparative Medicine, City of Hope National Medical Center, Duarte, CA, USA
To overcome poor pharmacokinetics and toxicity of triptolide
(TPL), a natural compound that exhibits potent anticancer ac-
tivities, we developed a novel antibody-drug conjugate (ADC)
to specifically deliver TPL to epidermal growth factor receptor
(EGFR)-overexpressing non-small cell lung cancer (NSCLC)
and others. The ADC (Cet-TPL) is made by conjugation of
TPL to lysine residues of cetuximab (Cet), a clinically available
anti-EGFR monoclonal antibody. Studies of antitumor efficacy
demonstrated that Cet-TPL drastically suppressed in vitro pro-
liferation and in vivo growth of these EGFR-overexpressing
cancers, including NSCLC A549 and H1299 cells and two pa-
tient-derived xenografts, and head and neck squamous carci-
noma UM-SCC6 cell, while it did not inhibit the proliferation
and growth of NSCLC H520 that rarely expresses EGFR.
Furthermore, immunofluorescence analysis revealed that Cet-
TPL was effectively internalized and transported into lyso-
somes of EGFR-overexpressing cells. Cet-TPL effectively led
to degradation of RNA polymerase II (Pol II) and demethyla-
tion of histone H3 lysines, and significantly induced apoptosis
in these EGFR-overexpressing cancers. Compared with TPL,
Cet, or their combination, Cet-TPL displayed higher target-
specific cytotoxicity against EGFR-expressing cancers and
much lower in vivo toxicity. In addition, Cet-TPL efficiently
suppressed the activated EGFR pathway in UM-SCC6 cancer
cells. Taken together, Cet-TPL represents a potent targeting
therapeutic agent against EGFR-overexpressing NSCLC
and others.
Many targeted therapies against kinases EGFR, anaplastic lymphoma
kinase (ALK), and c-Ros oncogene 1 receptor tyrosine kinase (ROS1)
have been developed with compelling clinical proofs of concept and
various survival benefits; however, treatment responses are typically
short-lived.2,3 Several KRAS-targeted therapies are under investiga-
tion, but none is currently approved for clinical use. Despite advances
in targeted therapies and immunotherapy, most lung cancers are still
incurable, and the 5-year survival rate for NSCLC patients remains
only 15%; therefore, new therapies are urgently needed.4
Triptolide (TPL) is a natural compound isolated from the Thunder
God Vine, Tripterygium wilfordii,5 which has been used in traditional
Chinese medicine to treat autoimmune disorders and inflammation,6
including lupus7 and rheumatoid arthritis.8 TPL has shown anti-
tumor activity in a variety of cancers, including lung cancer. TPL per-
turbs multiple signaling pathways, including NF-kB (nuclear factor
kappa-light-chain-enhancer of activated B cells),9 heat shock protein
70 (HSP70),10 BCL-2,11 and p53 pathways,12 and through these
signaling pathways TPL decreases cell proliferation and induces
apoptosis. Recently, a study reported that TPL covalently binds to hu-
man XPB, a subunit of the transcription factor TFIIH, and inhibits its
DNA-dependent ATPase activity that leads to the inhibition of RNA
polymerase II (Pol II)-mediated transcription,13,14 which may ac-
count for the majority of the known biological activities and molecu-
lar mechanism of action of TPL and be an explanation to many of its
therapeutic properties, including its robust and promising anticancer
properties. In lung cancer, TPL has been shown to sensitize cells to
TRAIL-induced apoptosis and enhance p53 activity.15 We previously
showed that treatment with TPL and its derivative led to decreased
lung cancer cell growth, invasion, and metastasis.16–18 Despite its
INTRODUCTION
Lung cancer is the leading cause of cancer-related deaths worldwide.1
The two predominant non-small cell lung cancer (NSCLC) histolog-
ical phenotypes are adenocarcinoma (ꢀ50%) and squamous cell car-
cinoma (ꢀ40%).1 KRAS and epidermal growth factor receptor
(EGFR) mutations are the most frequent oncogenic drivers discov-
ered in lung adenocarcinomas. For lung squamous cell carcinoma,
genes such as fibroblast growth factor receptors 1–3 (FGFR1–3)
and genes in the phosphoinositide 3-kinase (PI3K) pathway seem
to be more commonly mutated in lung squamous carcinoma.2,3
Received 16 April 2020; accepted 1 July 2020;
https://doi.org/10.1016/j.omto.2020.07.001.
4These authors contributed equally to this work.
Correspondence: Dan J. Raz, MD, MS, Division of Thoracic Surgery, City of Hope
National Medical Center, Duarte, CA, USA.
E-mail: draz@coh.org
Correspondence: David Horne, PhD, Department of Molecular Medicine, City of
Hope National Medical Center, Duarte, CA, USA.
E-mail: dhorne@coh.org
304
Molecular Therapy: Oncolytics Vol. 18 September 2020 ª 2020 The Author(s).
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

www.moleculartherapy.org
great promise as an anticancer drug in preclinical models, its clinical
use is limited by its toxicity and unfavorable pharmacokinetics,
including poor water solubility.19,20 Recently, TPL derivatives have
been developed in order to optimize its bioavailability and decrease
its toxicity. Minnelide, a TPL derivative, is currently in a phase II
study in metastatic pancreatic cancer and is given as a daily infusion
(ClinicalTrials.gov: NCT03117920).21 Another TPL derivative,
MRx102, has shown excellent anticancer effects in lung cancer and
leukemia in animal models with minimal toxicity.18,22
EGFR is highly expressed in the head and neck squamous carcinoma
UM-SCC6 cells and NSCLC A549 and H1299 cells, but not in
NSCLC H520 cells. The proliferation assays showed that TPL signif-
icantly suppressed the in vitro proliferation of all cancer cells in a
dosage-dependent manner (Figure S1), whereas Cet alone did not
inhibit the in vitro proliferation of A549 (Figure 2B), H1299 (Fig-
ure 2C), and H520 (Figure 2D), except for the proliferation of
UM-SCC6 cells (Figure 2E), indicating the EGFR signaling pathway
plays a crucial role only in cellular proliferation of UM-SCC6 cells.
Compared with the control (immunoglobulin G [IgG]) and Cet,
Cet-TPL displayed a dosage-dependent cytotoxic effect on all of
these EGFR-expressing cancer cells A549, H1299, and UM-SCC6,
except for H520, which does not express detectable EGFR (Figures
2B–2E), suggesting that Cet-TPL is specific for EGFR-expressing
cancer cells. Also, based on the ratio of half-maximal inhibitory con-
centration (IC50) of TPL to the conjugate of H520 (arbitrary index =
40) and that of H1299 (arbitrary index = 2), it may be concluded that
the conjugate shows high selectivity/affinity to EGFR-expressing
cancer cells.
Therapeutic antibodies have been proved an efficacious drug modal-
ity for their easy generation, specificity, and bio-durability. One strat-
egy to reduce the toxicity of TPL is to deliver it selectively to tumor
cells over their normal counterparts. EGFR is overexpressed on the
cell surface in most lung cancers,23 and it is also commonly overex-
pressed in many cancers, including head and neck, breast, colon,
ovarian, and cervical cancers, suggesting that anti-EGFR antibody
may be useful to deliver TPL to a variety of cancer types.24–27 Cetux-
imab (Cet) is an anti-EGFR antibody approved for treatment of head
and neck and colon cancers.28 Here, we report a novel antibody-drug
conjugate (ADC) that is built by conjugating TPL to the humanized
anti-EGFR monoclonal antibody (mAb) Cet (Cet-TPL), which may
specifically bind to the EGFR on cancer cells to deliver TPL. We
have tested this conjugate in lung cancer cell lines, as well as in several
mouse xenograft models of lung cancers and head and neck cancers.
We have found that Cet-TPL has potent anticancer activity against
EGFR-overexpressing cancer growth via the degradation of Pol II
subunit POLR2A of the TFIIH complex, one of the general transcrip-
tion factors for Pol II, and leads to multiple histone H3 lysine deme-
thylation. Compared with TPL, Cet, or their combinations, Cet-TPL
displays higher target-specific cytotoxicity against EGFR-expressing
cancers and much lower in vivo systemic toxicity. In addition, Cet-
TPL also efficiently suppresses the activated EGFR pathway in UM-
SCC6 head and neck squamous carcinoma cell. Taken together,
Cet-TPL represents a potential targeted therapeutic agent against
EGFR-overexpressing NSCLC and other cancers.
In addition, the cytotoxicities of TPL and Cet-TPL were also
examined on normal human bronchial epithelium cell line, BEAS-
2B, and human lung fibroblast cell line, MRC-5. TPL also signifi-
cantly inhibited in vitro proliferation of these two cell lines,
and the lung fibroblast MRC-5 cell was more resistant to TPL,
whereas Cet-TPL effectively suppressed the proliferation of BEAS-
2B cells that highly express EGFR, but rarely affected the prolifera-
tion of MRC5 in which EGFR is undetectable (Figure S2), further
indicating that Cet-TPL specifically targets all cells that highly
express EGFR.
In Vivo Suppression of Cet-TPL on EGFR-Overexpressing
Cancer Growth
We further investigated the specific antitumor efficacy of Cet-TPL
on xenografts of EGFR-expressing A549 cancer cells and two pa-
tient-derived xenografts (PDX1 and PDX2) derived from two lung
adenocarcinoma individuals in non-obese diabetic (NOD)/severe
combined immunodeficiency (SCID)/interleukin-2 receptor (IL-
2R) gamma null mice (NSG) mice. The data of tumor volume of sub-
cutaneous xenografts show that, compared with the groups treated
with Cet or TPL alone, only Cet-TPL significantly suppressed the
in vivo growth of NSCLC A549 and PDX1 xenografts (Figure 3A).
Although PDX2 and UM-SCC6 were sensitive to Cet and TPL,
compared with Cet or TPL alone, Cet-TPL (50 mg/kg) still displayed
a significantly stronger inhibition on the in vivo growth of these two
xenografts (Figure 3A). Consistently, the weights of cancer xeno-
grafts treated with Cet-TPL were significantly lower than those
treated with Cet and TPL alone. We also observed that a lower
dose of 25 mg/kg Cet-TPL also significantly suppressed the in vivo
growth of A549 xenografts and lung cancer PDX1 (Figure S3).
The dosage of 0.5 mg/kg TPL did not significantly suppress the
growth of A549 xenografts’ growth, and then other xenografts
were treated with 1.0 mg/kg TPL. In addition, we also tested a com-
bination of free Cet (equal to Cet control) plus TPL (the amount of
RESULTS
Characteristic Analysis of Cet-TPL Conjugates
Schematic of chemical conjugation of Cet with TPL is shown in Fig-
ures 1A and 1B. Figure 1C shows results of a sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) of Cet and Cet-TPL.
The samples were loaded with Laemmli sample buffer with or without
2-mercaptoethanol (2-ME) as marked. Figure 1D shows the mass re-
sults of the fast protein liquid chromatography (FPLC) purified Cet-
TPL after deglycosylation and reduction into the heavy chain and the
light chain. Based on the relative abundance of the mass peaks, an
average of about 5.5 TPLs per Cet was calculated.
In Vitro Cytotoxicity of Cet-TPL to EGFR-Overexpressing Cancer
Cells
To examine the antitumor efficacy of Cet-TPL, we first examined its
cytotoxicity to EGFR-expressing cancer cells compared with free
TPL. As shown in Figure 2A, western blot analysis reveals that
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Figure 1. Synthesis and Physical and Chemical Characteristics of Cet-TPL
(A) Schematic of chemical synthesis of triptolide (TPL)-NHS from TPL. (B) Schematic of chemical conjugation of cetuximab (Cet) with TPL-NHS and the formation Cet-TPL
conjugate. (C) SDS-PAGE gel for Cet (W), Cet-TPL conjugate (P), loaded with Laemmli sample buffer without (w/o 2-ME) or with 2-mercaptoethanol (w/2-ME) as marked. (D)
The mass spectrum of Cet (deglycosylated and reduced) in the full spectrum (upper diagram), for the light chain of the antibody (middle diagram), and for the heavy chain of
the antibody (lower diagram). An average of about 5.5 TPLs per Cet was observed. P, Cet-TPL conjugates purified by FPLC; W, Cet.
TPL is close to the amount of conjugated TPL on Cet-TPL) on the
in vivo growth of A549 xenografts; the outcomes of the experiment
indicated that the combination did not significantly suppress the
in vivo growth of A549 xenografts (Figure S4). Moreover, we also
examined whether Cet-TPL nonspecifically suppressed the in vivo
growth of xenografts generated by H520 that does not express
EGFR, and it turned out that Cet-TPL showed no inhibitory effects
on the in vivo growth of H520. In all, the data of the above experi-
ments clearly demonstrate that Cet-TPL specifically suppresses
EGFR-expressing cancer xenografts.
TPL in mice were recorded and compared with that of Cet-TPL.
During the treatment, signs of side effects, such as decreased activ-
ity, ruffled fur, decreased appetite, increased stool frequency and
diarrhea, weight loss, and death first occurred on the mice admin-
istered with TPL, and no abnormal symptoms of toxicity were
observed in mice administrated with Cet-TPL and other regimens.
It should be noted that the administration of these regimens to tu-
mor-bearing mice did not last longer, because the experiment was
terminated as soon as a mouse exhibited the above-abnormal
symptoms, especially weight loss and death. Compared with
mice given other regimens, the weight of mice administered with
TPL was slightly decreased at the end of the experiment (Fig-
ure S5), and 4 out of 32 mice died of TPL treatment, whereas
no mouse was dead in groups treated with Cet-TPL and other
The toxicity of TPL limits its clinical use; Cet-TPL is developed to
decrease systemic toxicity of TPL through specifically delivering
TPL to cancer cells. Therefore, the adverse effects or toxicity of
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Figure 2. In Vitro Cytotoxicity of Cet-TPL to EGFR-Overexpressing Cancer Cells
(A) Western blot analysis of EGFR in NSCLC cell lines A549, H1299, H520, PDX1, and PDX2 of human NSCLC. (B–E) A bar graph depicting the inhibitory effect of IgGs, Cet,
and Cet-TPL (conjugate) on in vitro cell proliferation for 72 h of (B) A549, (C) H1299, (D) H520 cells, and (E) UM-SCC6 (SCC6). The data are the average of triplicate ex-
periments; *p < 0.05 compared with the untreated parent cells.
regimens. Consideration of the amount of TPL showed it was
about 2-fold of the calculated amount of conjugated TPL on
Cet-TPL conjugate used in the study; therefore, compared with
TPL, Cet-TPL has stronger in vivo antitumor effects but much
less systemic toxicity in mice.
vealed, compared with these tissues treated with other regimens, the
percentage of Ki-67-positive cancer cells was drastically decreased in
EGFR-expressing xenograft tissues treated with Cet-TPL (Fig-
ure 4D), which was consistent with the in vitro data of cytotoxic as-
says, indicating the in vivo growth of EGFR-expressing cancers was
specifically significantly inhibited by the Cet-TPL. Because
compared with other cancer cells A549 cancer cells are less sensitive
to both Cet and TPL, we compared the apoptosis induced by these
treatments in A549 xenografts via staining and qualifying the
cleaved caspase-3 protein, and the analysis shows that Cet-TPL
induced significantly more apoptosis than Cet and TPL alone (Fig-
ure 4E), and quantitative analysis showed that the percentage of
cleaved caspase-3-positive cancer cells was much higher in A549
xenograft tissues treated with Cet-TPL than others (Figure 4F), indi-
cating Cet-TPL treatment induced significant in vivo apoptosis in
A549 xenografts.
Cet-TPL Significantly Decreased Ki-67-Positive Cancer Cells
and Induced More Apoptosis in EGFR-Overexpressing Cancer
Xenografts
The Ki-67 protein, a marker used to reflect cellular proliferation, was
analyzed in these cancer tissues by immunohistochemistry (IHC)
analysis. As shown in Figure 4, compared with the control (vehicle),
TPL, and Cet alone, the Cet-TPL dramatically decreased the Ki-67-
positive cancer cells in A549 xenografts (Figure 4A) and PDX1 (Fig-
ure 4B), whereas it did not decrease Ki-67-positive cancer cells in
H520 cells (Figure 4C). Quantitative analysis of Ki-67 IHC stains re-
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Figure 3. Treatment of EGFR-Overexpressing Cancer
Xenografts with Cet-TPL Significantly Suppressed In Vivo
Tumor Growth
(A) A line graph depicting the growth curves of A549, PDX1,
PDX2, and UM-SCC6 (SCC6) xenografts treated with vehicle
(IgG), TPL, Cet, or Cet-TPL (conjugate). Data are presented as
mean SD of eight mice (*p < 0.05, compared with vehicle). (B)
Bar graphs of the weights of A549, PDX1, PDX2, and SCC6
xenografts in mice treated with Cet (50 mg/kg), TPL (0.5 mg/kg
for A549 xenografts and 1 mg/kg for others), or Cet-TPL (50 mg/
kg); data are presented as mean SD; *p < 0.05, **p < 0.01, as
compared with vehicle/TPL. (C) The in vivo growth curves (left
panel) and weights of H520 xenografts (right panel) treated with
vehicle, Cet (50 mg/kg), and Cet-TPL (50 mg/kg).
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Figure 4. Immunohistochemistry Analysis of Proliferation Marker Ki-67 in Cancer Xenografts Treated with Cet-TPL
(A–C) Ki-67 staining in (A) A549 xenografts, (B) PDX1, (C) H520 xenografts treated with vehicle (VE), TPL (TR), Cet (CE), and Cet-TPL conjugate (CT). (D) Quantitative analysis
of the percentage of Ki-67-positive cancer cells in these xenograft tissues. (E) Apoptotic marker cleaved caspase-3 IHC staining in A549 xenografts treated with Cet-TPL. (F)
Quantitative analysis of the percentage of cleaved caspase 3-positive cancer cells in A549 xenograft tissues treated with Cet-TPL and others. Original magnification: ꢁ200;
red bar: 60 mm.*p < 0.05, **p < 0.01, compared with vehicle.
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Figure 5. Internalization and Distribution of Cet-TPL
in EGFR-Expressing Cancer Cells and Xenografts
(A) Immunofluorescence of an Alexa red fluorescein (red)-
labeled Cet-TPL conjugate with LysoTracker Green (green)
to localize Cet-TPL and lysosome in A549 cells (left panel)
and H520 cells (right panel); the arrows show the colocal-
ization of red (Cet-TPL) and green (lysosome) stains in A549
cells. (B) The in vivo distribution of Cet-TPL in A549 xeno-
graft tissues stained by HRP-labeled anti-human IgG. Left
panel: xenograft treated with TPL; right panel: xenograft
treated with Cet-TPL.
in vivo distribution of Cet-TPL in A549 xeno-
grafts by staining tissues with horseradish perox-
idase (HRP)-labeled anti-human IgG. IHC anal-
ysis shows that Cet-TPL (brown staining) was
indeed accumulated in the xenograft tissues (Fig-
ure 5B). Therefore, the above data indicate that
Cet-TPL can be delivered and specifically inter-
nalized and processed by EGFR-expressing can-
cer cells.
Cet-TPL Efficiently Suppresses Pol II and
Induced Apoptosis in Cancer Cells
It was reported that Pol II is a target that domi-
nantly mediates the biological activity of
TPL.13 Therefore, we examined the impact of
Cet-TPL on Pol II in comparison with that of
TPL. After cancer cells were incubated with
free TPL for 6 h, a dosage-dependent decrease
of both total and phosphorylated RNA polymerase II largest sub-
unit (RPB1, herein Pol II) was induced in both A549 (Figure 6A,
left panel) and H1299 (Figure 6B, left panel) cancer cells; at this
time point a decrease of these two proteins was not observed in
these cancer cells incubated with Cet-TPL (data no shown), while
at 18 h post-incubation, a dosage-dependent dramatic decrease of
both total and phosphorylated Pol II was observed in both A549
(Figure 6A, right panel) and H1299 (Figure 6B, right panel) cancer
cells, indicating that similar to the free TPL, Cet-TPL can effi-
ciently target Pol II of cancer cells in vitro.
Internalization and Distribution of Cet-TPL in Cancer Cells and
Xenografts
We then explored the mechanism of action of Cet-TPL by first con-
firming its binding and internalization in EGFR-expressing cancer
cells. As expected, Cet-TPL binds to its target EGFR to form a Cet-
TPL-EGFR complex, leading to endocytosis of the complex. The
internalized complex undergoes lysosomal processing, and the cyto-
toxic TPL is released inside of cells. The released free TPL binds to
its target, leading to cell death. We first examined whether Cet-TPL
normally bound to EGFR on the surface of cancer cells and then
was efficiently internalized. Using an Alexa red fluorescein (red)-
labeled Cet-TPL with LysoTracker Green (green) to show lysosome
in living cells, we tracked the binding and internalization of Cet-
TPL in EGFR-expressing A549 and EGFR-negative H520 cells after
it was incubated with cells for 6 h and then was washed off. Immuno-
fluorescence analysis showed that the red fluorescence of Cet-TPL can
be detected on both cell membrane and cytoplasm, and colocalized
with LysoTracker Green in EGFR-expressing A549 cells (Figure 5A,
left panel), whereas the red fluorescence of Cet-TPL was not observed
either on the membrane or in the cytoplasm of EGFR-negative H520
cells (Figure 5A, right panel), indicating that Cet-TPL can specifically
bind to the surface of EGFR-expressing cancer cells and then inter-
nalize and transport to lysosome. In addition, we also tracked the
Cet-TPL Efficiently Induces Apoptosis in Cancer Cells
In addition, we also compared apoptosis induced by free TPL and
Cet-TPL. Treatment of TPL can promptly induce apoptosis that
was displayed by cleaved PARP protein in both A549 (Figure 7A)
and H1299 (Figure 7B) cells at a dosage-dependent manner as
early as cancer cells were incubated with TPL for about 6 h.
Although the apoptosis induced by Cet-TPL was delayed in com-
parison with that of TPL, and pronounced apoptosis induced by
Cet-TPL was observed in A549 (Figure 7C) and H1299 cells (Fig-
ure 7D) after the cancer cells were incubated with Cet-TPL for 18
h, indicating the time course of induced apoptosis was consistent
with that of inhibition and degradation of Pol II.
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high dosage and lack of water solubility.19,20 ADCs were initially
developed to selectively deliver cytotoxic agents to cancer cells
through specific binding of an antibody to cancer-selective tar-
gets.29,30 So far, three ADCs, gemtuzumab ozogamicin, brentuximab
vedotin, and inotuzumab ozogamicin, have been approved for hema-
tologic cancers, and one ADC, trastuzumab emtansine, has been
approved to treat breast cancer; approximately 80 ADCs are in clin-
ical development in nearly 600 clinical trials, and 2–3 novel ADCs are
likely to be approved within the next few years.29 Two ADCs inhibit-
ing topoisomerase I activity, trastuzumab deruxtecan targeting HER2
in breast and gastric cancers and sacituzumab govitecan targeting
Trop2 in breast and lung cancers, are being examined in clinic.31
The EGFR is a member of the ErbB family of cell surface tyrosine ki-
nases that regulate both cell proliferation and apoptosis via signal
transduction pathways.32,33 Overexpression of the EGFR gene has
been identified in between 40% and 89% of NSCLCs.23 EGFR is
also overexpressed in approximately 75% of head and neck cancers,24
75% of malignant gliomas,25 and 70% of colorectal cancers,26 and is
overexpressed in a number of other cancers, including renal cell,
bladder, and pancreatic cancer.27 The overexpression of EGFR has
been exploited to target tumor cells selectively by small molecular in-
hibitor and anti-EGFR antibody to block the downstream signaling
pathway in some cancers, including colon and head and neck cancers.
Cet, a recombinant chimeric monoclonal IgG1 antibody as a EGFR
inhibitor, is approved for the first-line treatment in combination
with chemotherapy or as a single agent in patients who have failed
or are intolerant to chemotherapy with EGFR-expressing, RAS
wild-type metastatic colorectal cancer.28,34,35 ADCs targeting EGFR
have also been reported to target EGFR-expressing cancers
recently.36,37 In this study, we used Cet, an approved anti-EGFR anti-
body, as a vehicle to specifically deliver TPL to EGFR-expressing lung
cancers and others.
Figure 6. Suppression of Cet-TPL on RNA Polymerase II in Cancer Cells
(A and B) Western blot analysis of the total and phosphorylated Pol II in (A) A549 and
(B) H1299 cancer cells treated with TPL (nM) for 6 h (left panels) and Cet-TPL (mg/
mL) for 18 h, respectively.
Cet-TPL Efficiently Suppresses Multiple Histone H3 Lysine
Methylations and EGFR Signaling Pathways in Cancer Cells
We previously found that TPL modified global epigenetic changes of
histone H3 that were also associated with induced apoptosis in lung
cancer cells.16 Therefore, we also examined the inhibitory efficacy
of TPL and Cet-TPL on global epigenetic change in multiple histone
H3 lysine dimethylations in NSCLC cell lines. As shown in Figure 8,
after being treated with Cet-TPL for 18 h, global demethylations of
H3K4, H3K9, H3K27, H3K36, and H3K79 were dramatically sup-
pressed by Cet-TPL in a dosage-dependent manner in both A549
(Figure 8A) and H1299 cells (Figure 8B), which suggests that suppres-
sion on these modifications of H3 may also contribute to the
apoptosis induced by Cet-TPL in cancer cells.
Lastly, we also examined the effect of Cet-TPL on EGFR activation of
UM-SCC6 xenografts in which the EGFR signaling pathway may play
an important role in carcinogenesis and growth. As shown in Fig-
ure 8C, like Cet, Cet-TPL efficiently decreased phosphorylated
EGFR in xenograft tissues, indicating that Cet-TPL may function as
a bi-functional agent that can target Pol II and EGFR signaling path-
ways in these cancers with an activated EGFR signaling pathway.
It is well-known that an efficient ADC requires optimization of mul-
tiple parameters, including antigens, antibodies, linkers, payloads,
and the payload-linker linkage.38,39 In general, interchain disulfide
bridges and surface-exposed lysines are the most currently used res-
idues on the antibody for conjugation to cytotoxic payloads.40 Theo-
retically, the linkage of cytotoxic payloads to the surface-exposed
lysine of mAbs occurs after conjugation of ꢀ40 lysine residues on
both heavy and light chain of mAbs, and it results in 0–8 cytotoxic
payload linkages per antibody and heterogeneity with about one
million different ADC species.38–40 By controlling the ratio of TPL-
NHS to antibody, we got Cet-TPL with various drug-to-antibody ra-
tios (DARs) of 0.4, 2, 4, and 6 (calculated based on mass spectrum re-
sults; data not shown). We first characterized and optimized the DAR
of Cet-TPL and examined the efficacy of Cet-TPL with various DARs.
We found that DAR <2 did not work as efficiently as these conjugates
with a DAR from 4 to 6 (Figure S6), so we optimized our synthesis of
Cet-TPL with DARs of 4–6, and the average of DAR is about 5 for the
following study. We did not increase the DAR further because of a
study suggesting that antibody conjugates with a DAR ranging
from 2 to 6 have a better therapeutic index, less liver accumulation,
DISCUSSION
In this study, we have demonstrated that Cet-TPL, a novel ADC drug
that is built by conjugating TPL to the humanized anti-EGFR mAb
Cet (Cet-TPL), can specifically deliver TPL to EGFR-overexpressing
cancer cells and verified the in vivo-specific and potent antitumor ef-
ficacy of Cet-TPL in lung cancer and head and neck cancer models.
We believe this agent is likely to also be effective in a variety of other
EGFR-expressing cancers.
TPL has been shown to inhibit cell growth and lead to cell death in
many cancer cell lines at low dosages. The major obstacles for TPL
to becoming a clinically useful drug include its general toxicity at
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treatment.21 We also have shown that treatment with TPL and its de-
rivative leads to decreased lung cancer cell proliferation, invasion, and
metastasis of lung cancer.16–18 At the cellular level, TPL shows strong
antiproliferative activity, inhibiting the proliferation of all 60 US Na-
tional Cancer Institute cancer cell lines with IC50 values in the low
nanomolar range (average IC50 = 12 nM).13 In this study, we also
examined its effect on several lung cancer cells and found the IC50
of TPL to some cancer cells is as low as 6.25 nM. At the molecular
level, TPL was shown to perturb multiple signaling pathways,
including NF-kB,9 HSP70,10 BCL-2,11 and p53 pathways.12 In all,
TPL is a potential and ideal cytotoxic agent for cancer therapy.
The inhibition of Pol II-mediated transcription via TPL covalently
binding to and blocking its DNA-dependent ATPase activity XPB,
a subunit of the transcription factor TFIIH, has been confirmed to ac-
count for the majority of the known biological activities of TPL.13
Therefore, it is supposed that Cet-TPL conjugates would enter cancer
cells through EGFR, whereby the linker would undergo cleavage to
release TPL, allowing it to bind to XPB and block cell proliferation
or induce apoptosis. In agreement with this, we here have also
demonstrated, similar to TPL, that Cet-TPL effectively decreased
both total and phosphorylated Pol II, as well as the multiple lysine di-
methylation of histone H3, and induced apoptosis in the EGFR-pos-
itive cancer cells in a dosage-dependent manner.13,16 Notably, Cet-
TPL showed significant cytotoxicity against EGFR-positive cell lines,
but not EGFR-negative counterparts, in a dose-dependent manner.
Moreover, we showed that Cet-TPL significantly suppresses the
in vivo growth of EGFR-expressing cancer cells. Compared with
TPL, the effects of Cet-TPL were delayed but selective. The in vivo
growth of xenografts generated by A549 lung cancer cells and
PDX1 that are resistant to Cet and TPL were significantly suppressed
by Cet-TPL, whereas the in vivo growth of xenografts generated by
UM-SCC6 and PDX2 that were sensitive to both Cet and TPL were
also significantly suppressed by Cet-TPL compared with Cet or
TPL alone. In addition, Cet-TPL also efficiently suppresses the acti-
vated EGFR pathway in UM-SCC6 cancer cells. TPL is toxic at higher
doses and has relatively poor pharmacokinetics, and the lethal dose
(LD50) of TPL was reported as <1 mg/kg body weight in mice.43
We also observed a severe toxicity of TPL, although we did not
observe any significant toxicities (the most common being slight
weight loss, decreased appetite, and increased stool frequency and
diarrhea) in the mouse models treated with Cet-TPL. We envision
that conjugating TPL with Cet could also increase its selectivity for tu-
mor cells, thereby decreasing its toxicity. Compared with the in vitro
IC50 to lung cancer cells, the efficacy of Cet-TPL may be 1/50 of free
TPL, and we speculate that the low in vitro efficiency may be partially
caused by the delayed time course of cells’ response to Cet-TPL, and
an extended incubation of Cet-TPL with cancer cells may kill more
cancer cells and then further decrease the in vitro IC50 of Cet-TPL.
Of interest, compared with free TPL, the Cet-TPL has a better in vivo
anticancer effect. We believe the conjugate may change the stability
and distribution of TPL, because the IgG portion of the ADC is
important for maintaining a long half-life, binding to target, and
internalizing drug into tumor cells, and the longer half-life of the
Figure 7. Induced Apoptosis by Cet-TPL in Cancer Cells
(A and B) Western blot analysis of the apoptosis marker: cleaved PARP protein in
both (A) A549 and (B) H1299 treated with a given concentration of TPL (nM) for 3, 6,
and 18 h. (C and D) Western blot analysis of the cleaved PARP protein in both (C)
A549 and (D) H1299 treated with a given concentration of Cet-TPL (mg/mL) for 6, 18,
and 36 h.
and less clearance rates than these conjugates with a very high DAR
(ꢀ9–10).41 Binding and internalization, circulating stability, and
penetration of ADC are also essential to its cytotoxic effect of Cet-
TPL. In the study, we also clearly demonstrated that the conjugation
does not impact Cet’s binding affinity, and the Cet-TPL conjugate can
be effectively internalized in EGFR-expressing cancer cells. Also, bio-
distribution studies in xenograft tissues showed that the conjugate
could penetrate to the tumor microenvironment of subcutaneous xe-
nografts after intraperitoneal injection. Therefore, these data of
in vitro and in vivo experiments have convincingly indicated that
Cet-TPL can efficiently deliver TPL and transfer it into cancer cells.
TPL has been shown to have potent antiproliferative and cytotoxic ac-
tivities, and it is also effective against cancer in animal models.10,14,42
TPL and derivatives have entered human clinical trials for cancer
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mide (DCC) (108 mg, 0.52 mM) and N-hydroxysuccinimide (NHS)
(56 mg, 0.49 mM). After stirring overnight, the mixture was filtered
and concentrated under a vacuum. The residue was purified by silica
gel column chromatography (CH2Cl2/EtOAc, 3:1) to give TPL-NHS
(170 mg, 0.3 mM, 70%) as a white solid powder.
Cet (2 mg/mL) in 550 mL phosphate-buffered saline (PBS) in a 1,000-
mL glass bottle was added to TPL-NHS (80 mg, 20 eq.) in 8 mL N, N-
dimethylacetamide. The solution was gently stirred at room temper-
ature for 1 h. Tris buffer (1M [pH 8.0], 150 mL) was added to quench
the reaction and stirred for 30 min. Solution was concentrated by cen-
trifugal filters (Amicon Ultra-15) and purified by size-exclusion chro-
matography (HiLoad 26/600 Superdex 200). Concentrations of the
products were measured using A280. Purities were checked by
SDS-PAGE gels with or without reducing reagent 2-ME as indicated.
Cet control or purified Cet-TPL conjugates were treated with Rapid
PNGase F kit (New England Biolabs) to remove the N-linked glycans
and to reduce the antibody to light chain and heavy chain before in-
jecting on a quadrupole time-of-flight (Q-TOF) mass spectrometry
for easier observation. DARs were calculated by the relative abun-
dance of each individual peak from mass spectrum results.
Figure 8. Suppression of Cet-TPL on Multiple Histone H3 Lysine
Methylations and EGFR Signaling Pathways in Cancer Cells
(A and B) Western blot analysis of global demethylation of H3K4, H3K9, H3K27,
H3K36, and H3K79 in (A) A549 and (B) H1299 cells treated with Cet-TPL (mg/mL) for
18 h. (C) Western blot analysis of phosphorylated EGFR in xenografts of UM-SCC6
treated with vehicle, TPL, Cet, and Cet-TPL (conjugate); S1–S4 for four individual
samples. Veh, vehicle.
Cell Culture and Cytotoxicity Assay
Human lung cancer cell lines, A549 (p53 wild-type, K-Ras mutated),
H1299 (p53 null, N-Ras mutated), and H520 (p53 mutated), head
and neck squamous carcinoma cell line UM-SCC-6 (SCC6) cells,
and human normal fibroblast cell line MRC5 (CCL-171) were pur-
chased from American Type Culture Collection (ATCC). BEAS-2B
(CRL-9609; ATCC) immortalized human bronchial epithelial cell
line was derived from normal bronchial cells by immortalization
with an adenovirus 12-SV-40 hybrid virus, and it was cultured in
serum-free growth factor-supplemented BEBMTM (bronchial epithe-
lial cell growth basal medium, Lonza), as described previously.46 All
other cells were cultured in DMEM, Eagle’s minimal essential medium
(EMEM), or RPMI medium supplemented with 10% fetal bovine
serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin.
The cytotoxic effects of TPL, Cet, and Cet-TPL against cancer cell lines
were measured using the colorimetric cell proliferation cell counting
kit-8 (CCK-8). For proliferation assay, cells were seeded in 96-well
plates in four to six replicates at densities of 2.0 ꢁ 103 cells per well;
after 24 h, 3.125–100 mg/mL IgG, Cet, and Cet-TPL were added to
wells, respectively, and further incubated with cells for 72 h. Then
10 mL CCK-8 solution was added to each well and further developed
for 2 h. The absorbance values were detected at a wavelength of
450 nm using a Bio-Rad microplate reader. The cell viability was calcu-
lated by the optical density (OD) values of treated groups/OD values of
control groups (vehicle/PBS) ꢁ 100%. IgG also serves as a control for
Cet, and cell proliferation was monitored at 72 h using CCK-8. The
IC50 of TPL and Cet-TPL to each cell was also calculated accordingly.
IgG allows for greater absolute drug accumulation into tumors over
time.44 In all, compared with TPL, Cet, or their combination, Cet-
TPL displays higher target-specific cytotoxicity against EGFR-ex-
pressing cancers and much lower in vivo systemic toxicity.
Taken together, the present study provides convincing evidences that
Cet-TPL can effectively bind to EGFR and then is internalized inside
of cancer cells; finally it releases TPL to suppress Pol II in EGFR-ex-
pressing cancer cells, resulting in growth suppression of cancer cells.
We have verified that the novel Cet-TPL showed strong antitumor
properties against EGFR-positive cancers both in vitro and in vivo
without severe toxicity. Therefore, Cet-TPL represents a potent tar-
geting therapeutic agent against EGFR-overexpressing NSCLC and
other cancers.
MATERIALS AND METHODS
Synthesis and Characterization of Cet-TPL Conjugate
Schematic of chemical conjugation of Cet with TPL is shown in Fig-
ures 1A and 1B. After succinic anhydride (1,200 mg, 12 mM) and 4-
dimethylaminopyridine (DMAP) (72 mg, 0.6 mM) were added to a
solution of TPL (1,080 mg, 3 mM) in pyridine (6 mL), the mixture
was stirred overnight and diluted with ethyl acetate, then washed
with saturated copper sulfate, water, and brine, respectively. The
organic layers were dried over Na2SO4 and filtered. The filtrate was
concentrated and purified by silica gel column chromatography
(CH2Cl2/CH3OH, 15:1) to give the compound TPL succinate (TPS)
(1,100 mg, 2.4 mM, 80%) as a white solid powder.45
Xeno-transplantation and Treatment of Xenografts with Cet,
TPL, and Cet-TPL
TPS (200 mg, 0.44 mM) in dimethylformamide (DMF) (0.5 mL) and
dichloromethane (4 mL) was added to N,N0-dicyclohexylcarbodii-
This study was reviewed and approved by the Institutional Review
Board (IRB; #12299) of City of Hope National Medical Center. All
Molecular Therapy: Oncolytics Vol. 18 September 2020
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Molecular Therapy: Oncolytics
animal protocols were performed in the animal facility at City of
Hope National Medical Center in accordance with federal, local,
and institutional guidelines. All experiments using human material
were performed in accordance with all relevant guidelines and regu-
lations, and informed consent had been obtained from donors. NSG
mice (24–27 g, 6–8 weeks of age; Jackson Labs, Bar Harbor, ME, USA)
were used for xenograft experiment. Freshly harvested tumor speci-
mens from primary lung adenocarcinoma patients who underwent
surgical resection with curative intent without preoperative chemo-
radiotherapy were subcutaneously implanted in NSG mice to
generate PDX. When subcutaneous xenograft tumors reached
ꢀ1.5 cm3, they were serially passaged in NSG mice by subcutaneous
transplant (0.10–0.12 g, 2 ꢁ 2 mm) under general anesthesia.47 In the
study, two PDXs originating from two primary lung adenocarcinoma
individuals were used to examine the in vivo anticancer efficacy of the
conjugate. To establish xenografts of cancer cell lines, a suspension of
5 ꢁ 106 H1299, UM-SCC6 cancer cells in 0.1 mL RPMI 1640 was in-
jected into the subcutaneous dorsa of mice at the proximal midline.
When the tumor volume was about 90–110 mm3, mice were random-
ized into five or six treatment groups, and each group included seven
to eight mice. Also, mouse treatment was performed by intraperito-
neal injection of vehicle (PBS), 0.5 or 1.0 mg/kg TPL, 50 mg/kg
Cet, and 25 and 50 mg/kg Cet-TPL in <300 mL PBS twice/week for
about 2–3 weeks. The weight of the mice was recorded, and tumor
volumes were measured and calculated (0.5 ꢁ [long dimension] ꢁ
[short dimension]2) two to three times weekly. All animals were
euthanized before tumors reached 3,000 mm3 or showed signs of
reduced appetize, weight loss, or impending ulceration. At the end
of the experiment, mice were euthanized and xenograft tissues were
removed and weighted. Data are presented as average SD (*p <
0.05, compared with vehicle controls).
described previously.48,49 Data were presented as the average of per-
centages of three to four slides.
Cet-TPL was labeled Alexa red fluorescein (red), and the labeled Cet-
TPL and LysoTracker Green (green) to show lysosome in living cells
were used to track the binding and internalization of Cet-TPL in can-
cer cells by immunofluorescence analysis as previously reported.50 In
brief, Alexa red fluorescein-labeled Cet-TPL (1 mg/mL) was incubated
with cells in culture medium for 2 h, and then cells were washed with
PBS; after culturing for an additional 4 h, 100 nM LysoTracker Green
DND-26 (Cat#: 8783S; Cell Signaling) was added, and cells with fluo-
rescent staining of Alexa red and DND-26 green were imaged by fluo-
rescence microscope for 5–10 min.
Statistical Analysis
All experiments were performed in duplicates or triplicates and
repeated at least two times in each experiment. Two group compari-
sons were analyzed for variation and significance using a Student’s t
test or Pearson c2 test. All data shown are mean SD. Statistical sig-
nificance was set at p < 0.05.
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.
1016/j.omto.2020.07.001.
AUTHOR CONTRIBUTIONS
D.J.R. and D.H. conceived the study and revised the manuscript. K.Z.
and Y.M. designed and characterized the conjugate, analyzed and in-
terpreted data, and wrote and revised the manuscript. K.Z., Y.M., and
T.S. performed synthesis, cytotoxic assay, and western blot. Y.G. and
J.W. did xenograft experiments. R.P.P., M.L., and W.L. provided tech-
nical and material support. All authors read and approved the final
version of the manuscript.
Western Blot, IHC, and Immunofluorescence
The antibodies against Actin (catalog number [Cat#]: SC-47778),
total Pol II (Cat#: SC-55492), and phosphorylated Pol II (Cat#:
SC-47701) for western blot analysis were purchased from Santa
Cruz Biotechnology. The antibody against poly (ADP-ribose) poly-
merase (PARP; Cat#: PA5-77850) was purchased from Invitrogen.
The antibodies against total EGFR (Cat#: 4267S) and phosphorylated
EGFR (Cat#: 2234S), histone 3 (Cat#: 4499S), demethylation of H3K4
(HeK4me2, Cat#: 9725S), H3K9me2 (Cat#: 4658S), H3K27me2
(Cat#: 9728P), and H3K36me2 (Cat#: 2901S) were purchased from
Cell Signaling Technology.
CONFLICTS OF INTEREST
D.J.R., D.H., K.Z., and Y.M. have filed a patent based on the present
work. The other authors have no conflicts of interest to disclose.
ACKNOWLEDGMENTS
This study is supported by Stop Cancer and NIH grants
5K12CA001727-20 and P30CA33572 through the use of several
City of Hope Core Facilities. We acknowledge the generous support
of the Baum Family Foundation in support of this research. The au-
thors thank Aimin Li and Michael Lewallen at the pathology core for
IHC analysis.
A small proportion from each xenograft or PDX tissue was fixed with
4% formalin for 24 h and embedded in paraffin for subsequent histol-
ogy study. IHC analysis was applied to analyze the proliferation and
apoptosis marker proteins in A549 xenograft and PDX tissues using
anti-Ki-67 mAb (Cat#: M7240; Dako) and anti-cleave caspase-3 anti-
body (Cat#: NB100-56113; Novusbio). For quantitative analysis of
IHC staining, four regions of each slide were selected randomly; the
positively stained cancer cells were counted, and the percentage of
positively stained cancer cells to total cancer cells was calculated as
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