Bladder cancer therapy using a conformationally fluid tumoricidal peptide complex

Abstract

Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need.

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ARTICLE
Bladder cancer therapy using a conformationally
fluid tumoricidal peptide complex
Antonín Brisuda1,7, James C. S. Ho 2,6,7, Pancham S. Kandiyal3, Justin T-Y. Ng4, Ines Ambite 2,
Daniel S. C. Butler 2, Jaromir Háček5, Murphy Lam Yim Wan2, Thi Hien Tran2, Aftab Nadeem2,
Tuan Hiep Tran2, Anna Hastings 3, Petter Storm2, Daniel L. Fortunati3, Parisa Esmaeili2, Hana Novotna1,
Jakub Horňák1,Y.G.Mu 4, K. H. Mok 3, Marek Babjuk1,8 & Catharina Svanborg 2,8 ✉
Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent
tumoricidal activity. Here we define a peptide-based molecular approach for targeting and
killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A
39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor
cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance mea-
surements and computational simulations reveal a lipid core surrounded by conformationally
fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase
I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points
of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim
analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells
and the tumor size is reduced but no drug-related side effects are detected (primary end-
points). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the
expression of cancer-related genes is inhibited (secondary endpoints). The results are
especially encouraging for bladder cancer, where therapeutic failures and high recurrence
rates create a great, unmet medical need.
https://doi.org/10.1038/s41467-021-23748-y OPEN
1Department of Urology, Motol University Hospital, 2nd Faculty of Medicine, Charles University Praha, Prague, Czech Republic. 2Division of Microbiology,
Immunology and Glycobiology, Department of Laboratory Medicine, Faculty of Medicine, Lund University, Lund, Sweden. 3Trinity Biomedical Sciences
Institute (TBSI), School of Biochemistry & Immunology, Trinity College Dublin, The University of Dublin, Dublin, Ireland. 4School of Biological Sciences,
Nanyang Technological University, Singapore, Singapore. 5Department of Pathology and Molecular Medicine, Motol University Hospital, 2nd Faculty of
Medicine, Charles University Praha, Prague, Czech Republic.
6
Present address: Centre for Biomimetic Sensor Science, Nanyang Technological University,
Singapore, Singapore.
7
These authors contributed equally: Antonín Brisuda, James C.S. Ho.
8
These authors jointly supervised this work: Marek Babjuk,
Catharina Svanborg. ✉email: catharina.svanborg@med.lu.se
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Targeted cancer therapies have made significant advances
but the lack of tumor specificity remains a significant
concern1,2. Few current therapies kill cancer cells without
harming healthy tissues, and severe side effects have become
accepted as a necessary price to pay for survival or cure. The
notion of successfully combining efficacy with increased tumor
selectivity is justly regarded with skepticism. Yet, a serendipitous
discovery has provided insights into mechanisms of tumor-
specific cell death, induced by unfolded polypeptide chains, which
acquire tumoricidal activity by forming fatty acid complexes3,4.
Extensive observations in tumor models and clinical studies have
further defined the protein–lipid complexes as an interesting class
of molecules with significant therapeutic potential5.
The findings are challenging, as protein unfolding and loss of
structural definition is associated with a gain of toxicity, due to
the formation of amorphous aggregates and amyloid fibrils6,7.
Native protein structure is often regarded as a prerequisite for
biological function, by epitope-specific interactions and molecular
fitness for a finite number of cellular targets. Yet, a lack of
structural definition may, in some cases, result in a gain of
function, in part by uncovering different conformations and
exposing peptide motifs that are unavailable in the native state8,9.
Such effects have been predicted for membrane perturbing α-
helices in antimicrobial peptides, where the ability to destabilize
lipid bilayers has been proposed to reside in the three-
dimensional conformation rather than the amino acid
sequence10.
Alpha-lactalbumin is crucial for the survival of lactating
mammals. In its native state, the protein serves as a substrate
specifier in the lactose-synthase complex11,defining the nutri-
tional content and fluidity of milk. Partially unfolded alpha-lac-
talbumin, in contrast, forms an oleic acid complex, named
HAMLET, with potent tumoricidal activity3,4,8,9. The HAMLET
complex kills a range of tumor cells with rapid kinetics and shows
therapeutic efficacy in animal models of colon cancer, glio-
blastoma, and bladder cancer12–15. Early, investigator-driven
clinical studies demonstrated that HAMLET is active topically,
against skin papilloma and induces tumor cell shedding into the
urine in patients with bladder cancer5,16.
This study presents a synthetic, peptide-based drug candidate
derived from alpha-lactalbumin, which reproduces the tumor-
icidal properties of HAMLET and allows for a full translation of
these findings into the clinic. Through complementary nuclear
magnetic resonance (NMR) analysis and computational model-
ing, the molecular basis for this “gain-of-function”is defined,
including the three-dimensional structural motifs that determine
fatty acid-binding efficiency and tumoricidal activity. The ther-
apeutic efficacy of the complex is demonstrated in patients with
non-muscle invasive bladder cancer (NMIBC), in a fully con-
trolled clinical trial.
Results
Peptide-specific tumoricidal activity. To understand the invol-
vement of specific peptide motifs in tumor cell death, we syn-
thesized the N-terminal alpha-helical domain (residues 1–39,
alpha1) or the beta-sheet (40–80, beta) domains of human alpha-
lactalbumin (Fig. 1a). The alpha1 peptide formed complexes with
oleate (alpha1–oleate, 1:5) and circular dichroism (CD) spectra
detected an increase in alpha-helical structure content in these
complexes (Fig. 1b). The beta–oleate complex remained structu-
rally unchanged (Fig. 1b). Alpha1–oleate triggered a rapid, dose-
dependent death response in human lung- and kidney carcinoma
cells and in murine bladder cancer cells (Fig. 1c, d). The
beta–oleate complex lacked tumoricidal activity and tumor cells
subjected to the naked alpha-helical peptides (35 μM) or oleate
(175 μM) controls were not tumoricidal (Fig. 1c, d and Supple-
mentary Fig. 1). The loss of cell viability was irreversible, as
shown after 10 days, by using colony assays (Fig. 1e and Sup-
plementary Fig. 1). Membrane blebbing occurred in tumor cells
within minutes of exposure to alpha1–oleate but the naked
peptide- and oleate controls were not active (Fig. 1f and Sup-
plementary Fig. 1). Rapid K+fluxes were recorded, further
defining the membrane response (Fig. 1g). Pretreatment of the
cells with Na+and K+flux inhibitors reduced cell death by
40–50%, linking the membrane response to tumor cell death
(Fig. 1h). The alpha1–oleate complex was rapidly internalized by
tumor cells and by TUNEL staining, alpha1–oleate was shown to
induce double-strand DNA breaks in the tumor cells, indicative
of apoptosis (Fig. 1i, j).
In a screen of proteins with membrane-integrating properties,
SAR1 was found to form tumoricidal complexes with oleic acid,
reproducing effects of alpha1–oleate (Supplementary Figs. 1 and
2). SAR1 is a membrane-integrating protein of the COPII
complex that induces membrane tubulation by insertion of its N-
terminal amphipathic α-helix17–19. The N-terminal alpha-helical
peptide (residues 1–23, sar1alpha) formed an oleate complex,
which efficiently killed tumor cells (Supplementary Figs. 1 and 2).
The naked peptide- and oleate controls were not active
(Supplementary Fig. 1). Sar1alpha–oleate triggered membrane
blebbing in tumor cells, rapid K+fluxes were recorded, and
tumor cell death was partially inhibited by Na+and K+flux
inhibitors, suggesting a similar mode of action of the two
complexes, despite low sequence homology (Supplementary
Fig. 2). The sar1beta–oleate complexes and naked peptide
controls did not trigger tumor cell death, however (Supplemen-
tary Figs. 1 and 2).
In preparation for the clinical trial, the safety of alpha1–oleate
was investigated in C57BL/6J mice carrying MB49-induced
bladder tumors13. Therapeutic efficacy in 100% of treated mice
compared to the sham group and a lack of toxicity was
demonstrated, providing the necessary background to plan the
clinical trials13.
Biomolecular NMR analysis of the peptide–oleate complexes.
1H NMR spectra of the alpha1–oleate and sar1alpha–oleate
complexes detected a shift from sharp signals for the naked
peptides to broad signals and poor chemical shift dispersion for
the oleate complexes (Fig. 2a, b), suggesting a conformational
change from a random-coil fast-exchange time regime to an
intermediate millisecond timescale. Broadening in the amide,
side-chain methyl and aromatic regions suggests that interactions
between fatty acids and peptides occur throughout the molecules.
Two-dimensional nuclear Overhauser effect spectroscopy (2D
NOESY) spectra identified non-covalent, relatively short through-
space interactions between the respective peptides and fatty acids.
Important nuclear Overhauser effects (NOEs) were detected
between the olefinic protons (5.23 ppm) of oleic acid and the Hα
and aromatic protons of alpha1 and between the sar1alpha aro-
matic region and the oleic acid olefinic protons (Fig. 2c, d). The
downfield chemical shift of amide protons observed between 7.6
and 8.8 ppm suggests the presence of secondary structure in
alpha1 and alpha1–oleate. Well-resolved signals obtained from
the one-dimensional 1H NMR spectra provided a stoichiometry
of 3.7 oleate molecules per alpha1 peptide. Chemical shift map-
ping revealed a cluster of residues with aliphatic side chains that
change upon the binding of oleate, providing further evidence of
interactions between peptides and fatty acids (Fig. 2e, f).
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Hydrodynamic volume measurements carried out with size-
exclusion high-performance liquid chromatography (SE-HPLC)
and diffusion-ordered NMR spectroscopy (DOSY) showed that
the alpha1–oleate complex R
H
was considerably larger (27.4 and
29.3 Å, respectively) than the naked peptides (16.1 Å from SE-
HPLC and 14.4 Å from DOSY) (Fig. 2g, h and Supplementary
Figs. 3 and 4). The distinctly larger R
2
values (transverse
relaxation rate) for the complex than those of alpha1 peptide
and human serum albumin (HSA) suggested slower millisecond
to microsecond exchange processes (Supplementary Table 1 and
Supplementary Figs. 5 and 6). Importantly, the R
2
values for the
complex were also different from oleate in an aqueous solution,
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suggesting that the dynamics of the complex were clearly different
from micelle/vesicle-like particles formed from oleic acid/oleate
with no peptide component.
Computational analyses of the peptide- and peptide–oleate
system. Computational simulations also pointed toward struc-
tural heterogeneity, showing that the naked peptides and
peptide–oleate complexes belonged to wide conformational
spaces with relatively deep basins (Fig. 3a, b). Representative
structures mapped to different free energy surface minima,
revealed prominent alpha-helical secondary structural elements
and a hydrophobic oleate core for the peptide–oleate complexes
(Fig. 3c, e). The peptide–oleates fold upon this core differently
from the naked peptides, which exhibit multiple local minima
(Fig. 3d, f, and Supplementary Tables 2 and 3). Naked alpha1
ensembles were characterized by various partially folded helix-
turn conformations, whereas naked sar1alpha ensembles exhib-
ited a mixture of the random coil, alpha-helical, and beta-sheet
structures.
A contact probability analysis revealed that the interactions
between alpha1 or sar1alpha and oleate were mainly hydro-
phobic, with a >0.9 contact probability with olefinic protons
(Supplementary Tables 4 and 5). The peptide–oleate complexes
displayed relatively wide and deep free energy minima basins,
suggesting that a multitude of confirmations would be equally
possible to visit (Fig. 3c, e). When combined with the R
2
relaxation rates, the possibility of multiple sampling of various
conformations within a short period of time provides an
argument that rather than targeting specific partners, these
alpha-helical complexes may potentially be interacting with
multiple putative binding partners available on cancer cell
surfaces20.
Based on these extensive investigations and the strong agreement
of the experimental aspects with the simulated predicted ensembles,
it was clear that the apparently unrelated peptides alpha1 and
sar1alpha can form complexes with shared structural characteristics,
involving a flexible peptide moiety and a fatty acid cluster. This
notion resonates with the sequence-function inconsistency among
certain antimicrobial amphipathic alpha helices, where peptides
with similar overall properties, such as hydrophobicity or charge,
can have dramatically different levels of activity10.
A placebo-controlled, randomized clinical trial of alpha1–oleate
in patients with NMIBC. NMIBC is common and despite current
treatment protocols, recurrence rates are high21,22. To address if
the therapeutic effects observed in the murine MB49 bladder
cancer model can be translated into the clinic, the investigational
product alpha1–oleate was produced under GMP conditions. The
alpha1–oleate complex was further subjected to formal toxicity
testing and the results have been published13. Toxicity for bladder
tissue was not detected at concentrations ranging from 1.7 to 17
mM13.
The clinical safety and therapeutic potential of alpha1–oleate
were tested in a single-center, placebo-controlled, double-blind
Phase I/II trial (EudraCT No: 2016-004269-14, ClinicalTrials.gov
NCT03560479, Supplementary Note 1, Supplementary Table 6).
Patients with suspected NMIBC were randomized 1/1 to receive
alpha1–oleate or placebo during a period of 22 days, prior to
endoscopic removal of the tumor by transurethral resection
(TURB), (Fig. 4a, b). Alpha1–oleate (1.7 mM) or placebo (PBS)
was administered intravesical on six occasions (30 mL, days 1, 3,
5, 8, 15, and 22). The placebo solution was identical in appearance
to the active treatment. Demographic data, medical history, and
vital signs did not differ between the treatment and placebo
groups (for details see Supplementary Table 7).
Primary study endpoints. Adverse events (AEs) were recorded
and coded according to MedDRA (version 21.1) with a safety
follow-up after 52 days (Supplementary Table 8). Procedure-
related AEs, such as dysuria and bacteriuria, occurred at a similar
rate in the treatment and placebo groups. AEs specific for the
treatment group were not detected, suggesting low toxicity of the
study medication (Fig. 4c). Furthermore, there was no evidence of
a toxic response in healthy tissue samples from patients treated
with alpha1–oleate, defined by histopathology or TUNEL
staining.
Tumor cell shedding and release of tumor cell clusters were
recorded. Cells with uroepithelial morphology were quantified in
urine at each visit, before instillation and about 2 h after the
instillation of alpha1–oleate or placebo. Alpha1–oleate triggered a
rapid increase in cell shedding compared to the pre-instillation
sample in all treated patients, at all visits (Fig. 5a–c and
Supplementary Fig. 7). In addition, tumor cell clusters were
released into the urine in the treatment group. The clusters were
relatively large and the presence of tumor stroma in some samples
supported their tumor origin (Fig. 5d–f). The cells shed in urine
were assigned a pathology score as per the Paris classification
(classes 1–6, urine cytology was a secondary endpoint). In the
treatment group, an increase in the Paris score was detected in
post-inoculation samples compared to pre-inoculation samples
(Fig. 5g). A low level of cell shedding in the placebo group was
attributed to the instillation procedure and changes in pathology
Fig. 1 Tumoricidal activity of two non-homologous alpha-helical peptide–oleate complexes. a Ribbon representation of the crystallographically
determined three-dimensional structure of human α-lactalbumin (PDB ID: 1B9O), indicating the alpha1 (blue), beta (green), and alpha2 (gray) domains.
The calcium ion is not shown. bFar-UV circular dichroism spectra of synthetic alpha1 peptide, beta peptide, and their respective peptide–oleate complexes.
c,dDeath response in human lung (A549), kidney (A498), and murine bladder (MB49) carcinoma cells, quantified as a reduction in ATP levels (c,P=
3.26E−5 for A549, 0.013 for A498 and 0.005 for MB49, alpha1–oleate compared to beta–oleate) or PrestoBlue fluorescence (d,P=0.007 for A549,
0.003 for A498 and 0.002 for MB49, alpha1–oleate compared to beta–oleate). Cells were treated with the alpha1–oleate complex (blue) or the beta–oleate
complex (green), (3 h, 35 μM, cell death compared to PBS controls). For controls exposed to the naked peptides or oleate alone, see Supplementary Fig. 1d.
eColony assay showing dose-dependent long-term effects of alpha1-oleate. A representative image is shown from two independent experiments. Scale
bar =5 mm. fAlpha1–oleate triggers rapid membrane blebbing in A549 lung carcinoma cells (35 μM, 10 min). Scale bar =10 μm. A representative image is
shown from three independent experiments. gK+efflux in A549 lung carcinoma cells exposed to alpha1–oleate and inhibition with BaCl
2
.hInhibition of
cell death by the ion flux inhibitors Amiloride and BaCl
2
(100 μM), measured by PrestoBlue fluorescence (P=0.031 for 21 μM+BaCl
2
, 0.005 for 21 μM+
Amiloride, 0.028 for 35 μM+BaCl
2,
and 0.014 for 35 μM+Amiloride, compared to no inhibitor). iDNA strand breaks detected by TUNEL staining in
alpha1–oleate-treated A549 lung carcinoma cells (n=50 cells per group). Scale bar =20 μm. jAlexaFluor568-labeled alpha1–oleate (red) is internalized
by A549 lung carcinoma cells. Nuclei are counterstained with DAPI (blue) (n=52 cells per group). Scale bar =10 μm. Data are presented as mean ± SEM
from three independent experiments, *P< 0.05, **P< 0.01, ***P< 0.001, analyzed by two-tailed unpaired t-test (c,d,h,j) and 2-way ANOVA using
Dunnett’s correction (i).
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score or shedding of cell clusters were not observed (Fig. 5a–g and
Supplementary Fig. 7).
The endoscopic appearance of the tumors was recorded at the
time of diagnosis and prior to surgery using a flexible cystoscope
with white light-band and narrow-band imaging. Sizes were
assessed by an experienced endourologist using fully opened
clamps of the flexible forceps and a measuring device close to the
tumor. Paired images from 39 patients were evaluated in a
blinded manner, by an independent NMIBC expert using a
simplified Delphi method23 addressing changes in lesion size,
superficial necrosis, and tissue vascularization. A reduction in
lesion size was detected in the treatment group (n=19, Fig. 5h, i).
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In 1 patient, paired pre-treatment and post-treatment images
could not be collected for technical reasons. No difference in
superficial necrosis or vascularization was observed and there was
no change in lesion size in the placebo group (n=20).
Secondary end points. Evidence of tumor cell apoptosis was
obtained by staining of tumor biopsies obtained at the time of
surgery. Biopsies were examined for evidence of treatment-
induced apoptosis, quantified by the TUNEL assay (Fig. 6a–c and
Supplementary Fig. 8). A significant increase in net mean fluor-
escence was detected in the treatment group, compared to pla-
cebo (see also Fig. 1). Staining was most intense adjacent to the
lumen suggesting that a gradient might be formed, from the
lumen towards the center of the tumor. TUNEL staining intensity
was significantly correlated to cell shedding and alpha1–oleate
uptake in individual patients (Fig. 6d) but not to the tumor grade.
In healthy tissue biopsies from the treated patients, TUNEL
staining was low. Tumors from the placebo group did not show
increased TUNEL staining, suggesting that tumor cell apoptosis
may be induced by the alpha1–oleate treatment (Fig. 6c).
The alpha1–oleate content of shed cells in urine was quantified
by immunohistochemistry, using alpha1-specific antibodies.
Alpha1-staining was detected in 70% of post-inoculation samples
in the treatment group (Fig. 6e, f and Supplementary Fig. 9).
Uptake correlated with cell shedding and cluster grade but not
with the tumor grade or stage (Supplementary Fig. 9).
The response to alpha1–oleate was further evaluated by RNA-
seq, using RNA from tumor biopsies and comparing the
treatment to the placebo group. A strong treatment effect was
detected (Fig. 7a–c and Supplementary Fig. 10). Cancer-related
genes accounted for about 80% of the significantly regulated
genes in the treated patients (cut off fold change > 2.0, P< 0.05),
confirming the effect of alpha1–oleate on the tumor environment.
Genes regulating tumor growth and invasion were inhibited and
Ras signaling was suppressed, consistent with known effects of
the complex on Ras family members24 (Fig. 7d and Supplemen-
tary Fig. 10). Bladder cancer genes were specifically regulated,
including metalloproteinases, solute carriers, WNT complex
constituents, and thrombospondin, which affects angiogenesis25
(Fig. 7e). Furthermore, Fatty Acid Desaturase 6 (FADS6) and
transcriptional activator CREB3L4 were affected, suggesting that
the tumors respond to the constituents of the alpha1–oleate
complex. FADS6 regulates oleate biosynthesis and CREB3L4 the
unfolded protein response to conformationally fluid proteins,
such as the alpha1 peptide. Interesting targets also included the
gap junction alpha1 protein, which was inhibited, potentially
promoting cell detachment (GJA1/CXA1 encoding Connexin 43,
Supplementary Fig. 10). No difference in tumor grade was
observed between the treatment or placebo groups, defined by
WHO 1973 and 2004/2016 criteria (Supplementary Table 9).
Data regarding two secondary end-points are not reported. As
this is an interim analysis, the long-term treatment effects will be
evaluated when the entire study has been completed. The urine
proteomics data set has not been fully analyzed.
Discussion
Bladder cancer is the fourth most common malignancy in the
United States and the fifth in Europe, with a prevalence of about
1/400026. Due to high recurrence rates and a lack of curative
therapies, “bladder cancer has the highest lifetime treatment costs
per patient of all cancers, followed by colorectal-, breast-, pros-
tate-, and lung cancer”27. More than 80% recur after complete
surgical removal of the first tumor and 15% progress to muscle-
invasive disease28. Intravesical chemotherapy and Bacillus
Calmette–Guérin (BCG) immunotherapy have limited efficacy
and significant side effects29,30. Systemic administration of PD-1
and PD-L1 inhibitors is considered only in BCG unresponsive
patients where the experience is limited. Therapeutic options are
also limited by the inadequate supply of immunotherapy and
chemotherapy drugs worldwide31. In this study, we identify
conformationally fluid peptide–fatty acid complexes as additional
tools in cancer therapy and show that intra-vesical inoculation of
alpha1–oleate is safe and effective in patients with bladder cancer.
The tumor response to alpha1–oleate was analyzed in-depth,
using cellular and molecular tools to detect changes induced by
the complex. Treatment triggered the shedding of cells and tissue
fragments into the urine and alpha1–oleate internalization by
tumor cells confirmed the affinity of the complex for the tumor.
Further analysis of tissue biopsies suggested a lasting effect of the
alpha1–oleate instillations, as several tumor samples showed a
gradient-like pattern of apoptosis, starting from the bladder
lumen. Dysfunctional apoptosis has been identified as a key to
tumor development, especially in environments where oncogenes
such as MYC drive tumor cell proliferation32. Numerous attempts
have been made to develop apoptosis-inducing therapeutics with
tumor selectivity, but this has proven challenging, probably due to
the heterogeneity of individual tumors as well as their intrinsic
resistance to activating cell death pathways. The ability of
alpha1–oleate to stimulate apoptosis in the majority of bladder
tumors is, therefore, encouraging and consistent with the
apparent lack of toxicity for bladder tissue.
RNA sequencing revealed profound molecular changes in
treated tissues, attributable to alpha1–oleate. Classical cancer gene
networks were strongly inhibited in the treated patients, com-
pared to the placebo group, including Ras, previously identified as
a target for HAMLET; the oleate complex formed by the alpha-
lactalbumin holoprotein24. HAMLET binds activated Ras at the
plasma membrane of tumor cells and inhibits the Ras signaling
pathway, in part through effects on b-Raf phosphorylation. Sig-
nificant effects on adaptive immunity were not detected and
innate immunity was largely inhibited, including granulocyte
activation pathways. Notably, genes involved in oleate metabo-
lism and the unfolded protein response were affected, possibly
reflecting a direct response to the constituents of the
alpha1–oleate complex. In addition, treatment inhibited GJA1,a
Fig. 2 Biomolecular NMR analysis of naked alpha1- and sar1alpha peptides and their oleate complexes. a, b One-dimensional 1H NMR spectra. The
naked alpha1- (a, black) and sar1alpha- (b, black) peptides assume an ensemble of structures that interconvert rapidly and are therefore seen as sharp
peaks. The alpha1–oleate (a, red) and sar1alpha–oleate complexes (b, red) show broader peaks. Arrows indicate the indole 1H signals arising from the three
Trp side chains present in the sar1alpha peptide. c,dTwo-dimensional 1H NOESY spectra of alpha1–oleate and sar1alpha–oleate complexes, showing
atomic-level proximities of the fatty acid to the respective peptide. The spectra highlight NOEs between the 9,10 olefinic protons (5.23 ppm) of oleic acid
with the Hαprotons and aromatic protons of the alpha1–oleate complex (c) and the sar1alpha–oleate complex (d). e,fTwo-dimensional 1H–13C
Heteronuclear Single Quantum Coherence (HSQC) spectra overlays of the alpha1 peptide (red) and alpha1-oleate complex (black). Chemical shift
perturbation is detected in the aromatic side-chain region and the imidazole ring protons (e, green circled regions), and in the aliphatic side chain regions
(f). gSize-exclusion HPLC (SE-HPLC) of the alpha1 peptide and the alpha1–oleate complex, mapped onto a standard calibration curve. hDiffusion-ordered
NMR spectroscopy (DOSY) of the alpha1 peptide, alpha1–oleate complex, human serum albumin (HSA), and oleate suspension.
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gap junction protein that has been proposed to promote cancer
development and metastasis33,34. The effect occurred specifically
in tumor tissue, potentially providing the alpha1–oleate complex
with a mechanism to trigger cell shedding, as observed here. It is
interesting to speculate that cell shedding may serve as a “tip-of
the iceberg”marker of the profound changes in tumor biology
that include activation of programmed cell death, transcriptional
reprogramming, and inhibition of tumor progression.
Fig. 3 Free energy surface analyses of the peptide- and peptide–oleate system. a,bSuperimposition of dihedral principal component analysis
(PCA) plots of alpha1 (black points) and alpha1–oleate (red points) systems (a), and sar1alpha (cyan points) and sar1alpha–oleate (magenta points)
systems (b). Principal component (PC)1 and PC2 represent the axes of the two greatest variances after mathematical transformation for dimension
reduction. c–fFree-energy surfaces as a function of the first two principal components for alpha1–oleate (c), naked alpha1 (d), sar1alpha–oleate (e), and
naked sar1alpha (f). The representative structures of peptides or peptide–oleate complexes, along with their respective local minima annotations, are
colored from the N termini (blue) to the C termini (orange/brown). The free-energy surface of the alpha1–oleate complex contains 2 minima basins, A1 and
B1, with A1 representing the major conformational ensemble. The free-energy surface of the sar1alpha–oleate complex contains 3 minima basins, A3, B3,
and C3 (with the A3 basin harboring the major structural ensemble), which are characterized by a prominent alpha-helical secondary structural element, as
shown from simulation calculated alpha-helical propensities. By contrast, the free-energy surface of the naked sar1alpha shows large structural
heterogeneity. Here, minima basins A4 and D4 are represented by helical structures, B4 by beta structure, and C4 and E4 by random coil structures.
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Fig. 4 Clinical study protocol, demographic data, and adverse events. a Study CONSORT diagram.bStudy protocol. After diagnosis and informed
consent, the subjects received intravesical instillations of either alpha1–oleate or placebo on six occasions during one month preceding a scheduled
transurethral resection (TURB). A safety follow-up was performed 52 days after the first instillation. cNumber of adverse events (AEs) in the active and
placebo groups. No drug-related adverse events were recorded. There were totally 29 AEs reported by 12 subjects in the active group and by 11 subjects in
the placebo group. None of the AEs were related to the investigational product. One AE was severe and two were moderate in the placebo group. The
active group had one moderate AE. Two subjects in the placebo group reported severe AE (SAEs). The AEs were evaluated descriptively, and the AE
profiles were similar between the placebo and the active groups.
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Alternative therapeutic tools are actively being developed and
tested in patients with NMIBC, particularly in patients with
disease recurrence after BCG treatment35–37. Device-assisted
hyperthermia was shown to increase the efficacy of intra-vesical
chemotherapy but treatment was accompanied by side effects,
reducing compliance38–40. An oncolytic-virus-based intra-vesical
therapy was recently reported to achieve a complete response in
53.4% of patients with BCG-unresponsive carcinoma in situ, in a
phase III trial41. The authors discuss the assessment of side
effects and the development of biomarkers to help select patients
suitable for this therapy. In patients with BCG unresponsive
disease, treated with systemic Pembrolizumab, a 41% response
rate was reported but side effects were prevalent, limiting com-
pliance (Keynote-676 trial35). The present study identifies
alpha1–oleate as an active drug candidate with low toxicity.
Further dose-finding clinical studies and adjuvant therapy pro-
tocols will be essential to define the therapeutic window of this
complex.
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Cancer cells are aggressive, outcompete healthy cells, and ruin
tissue integrity. It is generally assumed that treatments must be
equally aggressive and highly toxic substances are often used,
despite their lack of selectivity and the severe side effects that they
cause. The protein–lipid complexes studied here are attractive to
cancer cells, which actively internalized them, but end up being
killed. Healthy cells are less responsive and extensive toxicity
studies have failed to detect adverse effects in the bladder13. This
low toxicity was confirmed here, as no drug-related side effects
were observed in the treatment group. The results, therefore,
identify the alpha1–oleate treatment of NMIBC as an interesting
therapeutic option. In view of the low toxicity observed so far,
liberal intra-vesical administration in early-stage NMIBC might
be an interesting approach to postponing the introduction of
more toxic and invasive therapeutic options.
Methods
Clinical trial of alpha1–oleate in NMIBC
Trial design and patient population. This was a single-center, placebo-controlled,
double-blinded randomized Phase I/II interventional clinical trial of non-muscle
invasive bladder cancer taking place from May 21, 2018 to June 3, 2019. Subjects
diagnosed with non-muscle invasive bladder cancer and scheduled for transure-
thral surgery were included in the study. The study was approved by the State
Institute for Drug Control (SUKL) in the Czech Republic; number 273799/17-I and
the Ethics Committee of the Motol University Hospital; number EK-786/17
(ClinicalTrials.gov Identifier: NCT03560479). Patients gave their written informed
consent.
Peptide synthesis and alpha1–oleate complex generation. Peptide synthesis and the
preparation of the investigational product were performed under good manu-
facturing practice (GMP) conditions and the complex was diluted in phosphate-
buffered saline (PBS) to the final concentration (1.7 mM). The placebo group
received PBS (sodium chloride, potassium chloride, sodium- and potassium
phosphate, and water for injection), which was identical in appearance to the active
treatment.
Study protocol. Study subjects were randomized 1/1 and received intra-vesical
instillations (30 mL) of either alpha1–oleate (1.7 mM) or PBS on six occasions
during a period of 22 days (days 1, 3, 5, 8, 15, and 22). A safety follow-up was
included 52 days after the last instillation (EudraCT Number: 2016-004269-14 and
ClinicalTrials.gov NCT03560479). The complete Study Protocol is provided as
Supplementary Note 1 in the Supplementary Information file. The interim analysis
of the clinical trial presented here represents a complete evaluation of the Phase I/II
study, as per the original protocol. The protocol was later amended to include a 24-
month follow-up. At that time, it was decided to perform the initially planned final
analysis as an interim analysis, scheduled after all subjects had completed the 52-
day safety follow-up. The study underwent data lock and subsequent unblinding
was under third-party control. The primary objective of the trial was to evaluate the
safety of alpha1–oleate. No formal sample size calculation evaluating the power of
the trial has been performed. However, consideration regarding the sample size was
made based on a previous open study of HAMLET instillations in bladder cancer
patients16 and in the murine bladder cancer model14. For efficacy, the sample size
was based on analysis of change in tumor cells assessed before HAMLET
instillation and after 2 h. The mean fold increase of shed cells was 41.3 and the
standard deviation was 60.4 in 9 patients16. A sample size of 20 patients per group
was deemed suitable to achieve criterion for significance (alpha) 0.05 and power
90% using the paired samples 1-tailed t-test. The null hypothesis is H
0
: mean
change in cell shedding =0 and the alternative hypothesis is H
A
: mean change in
cell shedding > 0.
Demographic data, morbidity and health parameters as well as tumor
characteristics were recorded by the study physicians in the electronic Case Report
Form (eCRF) and closely monitored by an external monitor. Population
characteristics were evaluated by the study statistician. No significant differences
between the treatment and placebo groups were registered in terms of age, gender,
co-morbidity, or tumor parameters.
Primary endpoints. - Safety as AEs profile (time frame: from the signing of
informed consent (day 1) and until end of study (day 52)): Incidence of AEs and
classification in terms of severity, causality, and outcome.
-Efficacy as cell shedding (time frame: days 1–22): change in cell shedding into
urine (number of epithelial cells per mL of urine).
- Change from baseline in characteristics of papillary tumors (time Frame: prior
to treatment (baseline) and on day 30, in connection with scheduled surgery): the
bladder tumors are characterized by in vivo imaging during examination by
cystoscopy.
Secondary endpoints. - Histopathology scoring of the tumor using established
parameters for scoring of Grade and Stage/Invasiveness.
- Urine cytology examined before and after instillation, using the Paris scoring
system.
- Uptake of alpha1–oleate by tumor cells, defined by staining with specific
antibodies.
- Tissue apoptotic response to alpha1–oleate, defined by TUNEL staining.
- Tumor response to alpha1–oleate, defined by RNA sequence analysis.
- Proteomic analysis of markers in urine was not completed.
- Long-term effects of the study treatment have not been evaluated.
AEs profile. AEs were collected from the signing of the informed consent form until
the end of the study (FU1 Visit, day 52). All diagnoses, symptom(s), sign(s), or
finding(s) with a start date after the first dose of the study drug were recorded as
AEs or severe AEs (SAEs). (S)AEs related to the study procedure were coded
during the course of the trial according to MedDRA by preferred terms and pri-
mary system organ class. All AEs recorded during the course of the trial were
included in the subject data listings and an overall summary of the number
(percentage) of subjects with any treatment-emergent (S)AEs, premature dis-
continuations from the trial due to AEs, treatment-related AEs, and SAEs were
constructed. The number of subjects experiencing each type of adverse event was
tabulated regardless of the number of times each adverse event was reported by
each subject. The severity of each type of adverse event was also tabulated and
graded as the most severe recording for that adverse event.
Cell shedding. To quantify the shedding of cells and cell clusters into the urine,
samples were obtained from each patient prior to and after each instillation of
alpha1–oleate or placebo (Visit 1–Visit 6). Cell shedding was quantified by
counting the total number of epithelial cells in a unit of uncentrifuged urine under
light microscopy, using a hemocytometer chamber. Changes in cell shedding were
quantified at each visit, by comparing cell numbers in samples obtained before and
after each instillation. The cell clusters were scored based on the examination of
these samples by an experienced pathologist on a range of 0–2 where 0 =no
clusters and 2 =the highest number of clusters.
Fig. 5 Primary endpoints: shedding of tumor cells and reduction in tumor size following intra-vesical instillation of alpha1–oleate. a–cCell shedding
increased significantly after alpha1–oleate instillation. aScatterplot showing individual means of six visits per patient in the treatment group (n=20)
compared to patients receiving placebo (n=20). Line represents the median. bComparison of cell numbers in urine before (pre =white) and after (post
=black) alpha1–oleate inoculation on visits 1–6 showing increased cell numbers post-inoculation in the treatment group (n=20 patients per group, P=
0.0030 for visit 1, 0.0098 for visit 2, <0.0001 for visits 3 and 4, 0.0073 for visit 5 and 0.0336 for visit 6) but not in the placebo group. Data are presented
as mean ± SEM. cRepresentative images, illustrating the increase in cell shedding after alpha1–oleate instillation. Magnification =×400. Scale bar =50 μm.
d–fDifference in the shedding of tumor cell clusters between the treatment and placebo group. dScatterplot showing individual means of six visits per
patient in the treatment group compared to patients receiving placebo. Line represents the median. eIncreased numbers of cell clusters in post-inoculation
samples of patients receiving alpha1–oleate (n=20 patients per group, P=0.9743 for visit 1, 0.0212 for visit 2, <0.0001 for visits 3, 4, and 5, and 0.0005
for visit 6). Data are presented as mean ± SEM. fRepresentative images of cancer cell clusters after alpha1–oleate instillation. Magnification =×400. Scale
bar =50 μm. gParis grade of shed cells before or after alpha1–oleate instillation. An increase is observed in the treatment group (χ2test). hReduction in
tumor size in patients receiving alpha1–oleate treatment. Images were compared between the time of diagnosis and the time of TURB (P=0.04, χ2test for
trend compared to placebo, n=19 for treatment group and n=20 for placebo group). iExamples of cystoscopy photographs obtained by A.B. at the time
of diagnosis and after treatment at the time of TURB. Scale bars =5 mm. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001. The data were analyzed by
two-tailed unpaired Mann–Whitney U-test (a,d) or by repeated-measures two-way ANOVA with Sidak’s correction (b,e).
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Characteristics of papillary tumors, tumor size. To examine if the alpha1–oleate
treatment affects tumor size, all included subjects underwent outpatient cystoscopy
at Visit 0. Tumors were reexamined at Visit 7, prior to scheduled surgery. High-
quality photographs were collected endoscopically, using a flexible cystoscope
(Olympus) before being removed by TURB according to EAU Guideline
recommendations42. Changes in tumor size were evaluated intra-individually,
using paired images. The results were evaluated using a simplified Delphi
method23, by an independent NMIBC expert. Changes in lesion size, superficial
necrosis, and tissue vascularization were addressed in a blinded manner.
Histopathology scoring. Tumor biopsies, collected at the time of surgery were
evaluated by histopathology, using established parameters for scoring of Grade and
Stage/Invasiveness. Tissue samples were analyzed by a designated study uro-
pathologist. Both grading classifications (WHO 1973 and 2004/2016) were used43.
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Biopsies from healthy tissue areas distant from the tumor were collected for
comparison.
Urine cytology. Urine cells were centrifuged onto L-lysine-coated microscope slides
(Cytospin 3, Shandon) at 113×gfor 5 min, fixed and stored at room temperature
until further analyses. Urinary cytology was evaluated using the Paris System for
Reporting Urinary Cytology 201643,44 and defined as: 1. No diagnosis/unsatisfac-
tory. 2. Negative for high-grade urothelial carcinoma. 3. Atypical urothelial cells
present. 4. Suspicious for high-grade urothelial carcinoma. 5. High-grade urothelial
carcinoma. 6. Low-grade urothelial neoplasm. 7. Other positive for malignancies
and miscellaneous lesions.
Alpha1–oleate uptake. Alpha1–oleate uptake by tumor cells was quantified by
staining with specific antibodies. Cells on cytospin-slides were washed (Tris-buffer
saline TBS, 10 min), permeabilized (0.25% TritonX-100 in TBS, 20 min, room
temperature) and blocked (5% normal goat serum in TBS, 1 h, room temperature)
before the addition of rabbit polyclonal anti-human alpha-lactalbumin antibodies
(1:50 in 5% normal goat serum at 4 °C, overni ght, Mybiosource, Cat# MBS175270).
Slides were washed (TBS, 2 ×5 min) and stained with Alexa-568-labeled secondary
antibody (1:200, 1 h, room temperature, ThermoFisher). The nucleus was coun-
terstained using DRAQ5 (1:1000, 15 min) before a final wash (2 ×5 min in TBS).
Slides were mounted (Fluoromount aqueous mounting media), before capturing
images by laser scanning confocal microscopy (Carl Zeiss). Fluorescence intensity
was quantified by ImageJ and net fluorescence calculated after subtraction of the
secondary antibody background.
Apoptosis in tissue biopsies, TUNEL staining. DNA fragmentation was detected
using the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)
assay (Click-iT TUNEL Alexa Fluor 488 imaging assay kit, ThermoFisher). Tissue
sections were de-paraffinized with xylene followed by serial dehydration with
ethanol (100%, 95%, 75%, and 50%). Dehydrated sections were fixed (4% PFA, 15
min), permeabilized (DNase-free Protease K solution 20 µg/mL, 15 min) and
incubated with TUNEL reaction mixture containing TdT for 60 min at 37 °C. After
the TUNEL reaction, sections were incubated with Click-iT reaction mixture (30
min, 37 °C). Sections were counterstained with DAPI (1 µg/mL, 5min), mounted in
Fluoromount aqueous mounting media, and analyzed by fluorescence microscopy
(Zeiss). Fluorescence intensities were quantified by ImageJ and net mean fluores-
cence intensity calculated after subtraction of background fluorescence.
RNA sequence analysis of tissue biopsies. RNA was extracted from tissues
stabilized in RNAlater using the AllPrep DNA/RNA/miRNA Universal Kit. Dis-
ruption was in the TissueLyser system and CK28 Precellys tubes and by homo-
genization in the QIAshredder homogenizer. The quantity and quality of the RNA
samples were evaluated using NanoDrop and Agilent 2100 Bioanalyzer. RNA
samples were prepared by Illumina TruSeq Stranded mRNA Library Prep Kit
(20020594), and libraries were multiplexed and sequenced using NextSeq 500/550
High Output Kits (v2.5 2 × 75 Cycles) with an average of 22 million reads per
sample. Raw sequencing data were demultiplexed using bcl2fastq (version 2.18)
and RSEM (1.3) was used for abundance estimation using the human genome
release 37/Ensemble 75. Samples were thoroughly quality checked (QC) and
visualized using dimensionality reduction (i.e. PCA), MA-plots as well as RNA-seq
intrinsic biases (such as GC bias, transcriptome complexity, and alignment quality).
Differential expression analysis was performed using R (version 3.4) and the
packages limma and DESeq2. Fold changes were calculated by comparing tumors
in the treated to the placebo group. Relative expression levels were analyzed and
genes with an absolute fold change >2.0 and P< 0.05 were considered as differ-
entially expressed. Heat-maps were constructed using the Gitools 2.1.1 software.
Differentially expressed genes were functionally characterized using the Ingenuity
Pathway Analysis version 57662101 (IPA, Qiagen) software.
Statistical analysis. For efficacy, the sample size was based on analysis of change
in tumor cells assessed from a previous study16. A sample size of 20 patients per
group was deemed suitable to achieve criterion for significance (alpha) 0.05 and
power 90% using the paired samples 1-tailed t-test. The null hypothesis is H
0
: mean
change in cell shedding =0 and the alternative hypothesis is H
A
: mean change in
cell shedding > 0. The Gaussian distribution was determined by the D’agostino and
Pearson normality test. For data following a Gaussian distribution, student t-tests
were used. Other data sets were analyzed by Mann–Whitney U-test. Correlations
were determined by Spearman correlation. Kinetic data were analyzed using the
repeated measures two-way ANOVA test. All statistical analysis was done by using
Prism version 6.02 (GraphPad Software Inc.). Pvalues < 0.05 were considered
statistically significant. All images were created by the study team.
Molecular and cellular studies
Chemicals and antibodies. Sodium oleate (Sigma-Aldrich, Cat# O7501), Alexa-
Fluor568 protein labeling kit (Thermo Scientific, Cat# A10238), AlexaFluor488
protein labeling kit (Thermo Scientific, Cat# A10235), ATPlite (Perkin Elmer, Cat#
6016947), Presto Blue Cell Viability Assay (Invitrogen, Cat# A13262), Anti-peptide
antibodies (this study, produced by GeneCust), FluxOR Potassium ion channel
assay (Invitrogen, Cat# F20015), Barium chloride BaCl
2
(Sigma-Aldrich, Cat#
B0750), Amiloride (Sigma-Aldrich, Cat# A7410), Click-iT TUNEL Alexa Fluor 488
imaging assay kit (ThermoFisher Scientific Cat# C10245), DRAQ5 (Abcam, Cat#
ab108410), Fluoromount (Sigma-Aldrich, Cat# F4680), DNA/RNA/miRNA Uni-
versal Kit (Qiagen, Cat# 80224),
Peptide synthesis and complex generation. Peptides for in vitro and in vivo
experiments were synthesized using Fmoc solid-phase chemistry (Mimotopes,
Melbourne, Australia). For biotinylated peptides, an aminohexanoic acid (Ahx)
spacer was added to ensure adequate separation between the biotin and the peptide
moieties. A five-fold stoichiometric concentration of sodium oleate in phosphate-
buffered saline was prepared, followed by the addition of each respective peptide.
The more hydrophobic peptides were initially dissolved in DMSO, then transferred
to the oleate buffer. The sequences for the peptides are as follows:
Alpha1: Ac-KQFTKAELSQLLKDIDGYGGIALPELIATMFHTSGYDTQ-OH
Beta: Ac-IVENNESTEYGLFQISNKLWAKSSQVPQSRNIADISADKFLD
DD-OH
Sar1alpha: Ac-MAGWDIFGWFRDVLASLGLWNKH-OH
Sar1beta: Ac-DRLATLQPTWHPTSEELAIGNIKFTTFDLGGHI-OH
Cell lines and cell culture. Human lung carcinoma cells (A549, ATCC Cat# CCL-
185, RRID:CVCL_0023), human kidney carcinoma cells (A498, ATCC Cat# HTB-
44, RRID:CVCL_1056), and murine bladder carcinoma cells (MB49, RRID:
CVCL_7076, provided by Sara Mangsbo, Uppsala University, Sweden) were cul-
tured in RPMI-1640 with non-essential amino acids (1:100), 1 mM sodium pyr-
uvate, 50 μg/mL gentamicin, and 5–10% fetal calf serum (FCS) at 37 °C, 5% CO
2
.
Cell death assays. To quantify effects on cell viability, A549, A498, or MB49 cells
were seeded in 96-well plates (2 × 104/well, Tecan Group Ltd.), cultured overnight
at 37 °C, 5% CO
2
and incubated with peptide–oleate complexes in serum-free
Fig. 6 Apoptotic response to alpha1–oleate and cellular uptake by tumor cells. Apoptosis was quantified in tumor biopsies, using the TUNEL assay.
Arbitrary units were calculated after subtraction of background staining in TUNEL negative healthy tissue samples. aRepresentative image of TUNEL
staining (green =TUNEL, blue =DAPI) in tumor tissue from individual patients receiving alpha1–oleate instillations. Scale bars =200 μm. bRepresentative
images of TUNEL staining in tumor tissue from individual patients receiving placebo. Scale bars =200 μm. cScatter plot demonstrating elevated TUNEL
staining intensity in tumor biopsies from patients receiving alpha1–oleate instillations compared to placebo. TUNEL staining was not significantly altered in
healthy tissue biopsies from patients receiving alpha1–oleate instillations or placebo (n=40 tumors and 38 healthy biopsies, two data points were further
removed due to medical conditions from patients and confirmed by Grubbs’s outlier test) (two-tailed unpaired Mann–Whitney U-test). Line represents the
median. dCorrelation of TUNEL staining intensity with cell shedding (P=0.03, 95% CI 0.0220–0.6010) and alpha1–oleate uptake (P=0.01, 95% CI
0.0957–0.6461) (Spearman correlation, two-tailed, approximate P-value, n=20 for alpha1–o and n=19 for placebo). eRepresentative images of
alpha1–oleate (red) uptake with counter-stained nuclei (blue). Alpha1–oleate uptake by tumor cells was quantified by staining of shed cells in urine with
polyclonal anti-alpha1–oleate antibodies. Scale bars =20 μm. fScatterplots of cellular uptake in individual patients receiving alpha1–oleate. Each dot
represents the mean fluorescence intensity of six post-instillation samples per patient treated with alpha1–oleate. Comparison of alpha1–oleate uptake by
the cell in urine before (pre =white) and after (post =black) alpha1–oleate inoculation on visits 1-6 (repeated-measures two-way ANOVA with Sidak’s
correction, P=0.0049 for visit 1, 0.1913 for visit 2, 0.0067 for visit 3, 0.0025 for visit 4, 0.3807 for visit 5 and 0.0043 for visit 6, n=20 per group and
time point). Line represents the median and bars represent mean ± SEM.
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RPMI-1640 at 37 °C. FCS was added after 1h and cell viability was quantified after
3 h, by the Presto Blue fluorescence assay (Thermo Scientific) or ATP levels
(luminescence-based ATPlite kit), using a microplate reader (Infinite F200, Tecan).
The colony assay was used to define long-term effects on cell viability. MB49
cells were seeded in a 24-well tissue culture plate (1000 cells/well in 1 mL in RPMI
with FCS), allowed to adhere for 24 h, was hed in PBS and treated with
alpha1–oleate or sar1alpha–oleate (7, 21, or 35 μM in 1 mL of RPMI without FCS
for 1 h). After the addition of FCS (5%) the cells were incubated at 37°C in 5% CO
2
for 10 days or until visible colonies were present in the PBS control. Plates were
fixed in cold methanol, stained with hematoxylin. Staining intensity was quantified
using ImageJ.
TUNEL staining was performed using Click-iT TUNEL Alexa Fluor 488
Imaging Assay kit (Thermo Scientific). Briefly, the cells were fixed in 2%
paraformaldehyde for 15 min followed by permeabilization with 0.25% TritonX-
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100 for 20 min. The sections were incubated for 60 min at 37 °C with the TUNEL
reaction mixture, then counter-sta ined with DAPI (20 μg/mL) for 15 min. A
positive contro l slide (cells treated with DNase I for 30 min at 37 °C) was included
in each experiment. The TUNEL fluorescent intensity was quantified using Image J.
Membrane blebbing and ion fluxes. Membrane changes in lung carcinoma cells
were visualized by transmission light microscopy imaging. Cells were seeded on a
glass coverslip and allowed to partially adhere to the glass surface for 10 min at
room temperature, prior to exposure to the alpha1–oleate or sar1alpha–oleate
complexes. Changes in cell morphology were captured with LSM 510 META
confocal microscope (Carl Zeiss) using ×40 oil immersion objective. Naked pep-
tides and oleate were used as negative controls (Supplementary Fig. 1e).
K+fluxes were quantified using the FluxOR potassium ion channel assay
(Invitrogen). The influx of the indicator Ti+measures the opening of K+
channels45. Briefly, 20,000 adherent cells were incubated in loading buffer followed
by incubation with assay buffer and stimulus buffer for 60 min. For inhibition, cells
were pretreated with the K+channel inhibitor BaCl
2
(100 μM) followed by
treatment with alpha1–oleate (35 μM). Fluorescence was measured at 535 nm after
excitation at 485 nm using a fluorescence plate reader (TECAN infinite F200).
Structural analysis of the peptide–oleate complexes
CD spectroscopy. Far-ultraviolet (UV) CD spectra were collected on alpha1-, beta-,
sar1alpha-, and sar1beta-peptides with and without oleate at 25 °C using a Jasco
815 CD Spectropolarimeter. The peptides were dissolved in 50 mM sodium
phosphate buffer, pH 7.4, with 10% D
2
O, at a final concentration of 0.2 mg/mL.
Far-UV CD was performed from 185 to 260 nm for the samples without oleate and
from 200 to 260 nm for the samples with oleate and the background was sub-
tracted. The mean residue ellipticity (MRE), [θ], in deg cm2/dmol, was calculated as
described previously9.
Biomolecular NMR spectroscopy. The alpha1- and sar1alpha-naked peptide samples
were dissolved in 50 mM sodium phospha te buffer (pH 7.4, 90% H
2
O, 10% D
2
O),
and the peptide–oleate complexes were reconstituted from a lyophilisate of
phosphate-buffered saline. All experiments were carried out in the phase-sensitive
mode46. One-dimensional 1H, two-dimensional NOESY (Nuclear Overhauser
Effect Spectroscopy) and 1H–13C HSQC (Heteronuclear Single Quantum Corre-
lation) spectra were acquired on an Agilent Technologies 18.8T (800 MHz) DD2
Premium Compact spectrometer with a triple-resonance, 5 mm enhanced cold
probe. The 1H–13C HSQC spectra were collected at 20 °C with 16 scans, an initial
delay of 3.0 s, a 90° pulse width of 7.5 and 9.8 μs, and an acquisition time of 0.4 s
with broadband decoupling for alpha1 and sar1alpha peptide samples. For the
alpha1–oleate and sar1alpha–oleate complexes, we used a 90° pulse width of 12.80
and 13.30 μs and an acquisition time of 0.4 s. All acquisition parameters were kept
constant for all samples. Two-dimensional DPFGSE-NOESY (Double Pulse Field
Gradient Spin Echo-NOESY) pulse sequences were used to acquire data at 20 °C
with 16 scans, with an optimized mixing time of 300 ms for the alpha1 and
alpha1–oleate complexes and a delay period of 1.5 s. For the sar1alpha–oleate
complex, water-gate NOESY was used with 12 scans, with a mixing time of 150 ms.
A trace amount of TSP was added to serve as a chemical shift reference. Each 2D
HSQC spectrum consisted of 4 K complex points in the acquisition dimension and
512 complex points in the indirect dimension. For the NOESY spectra, 4 K com-
plex points were used in the acquisition dimension and 1 K complex points in the
indirect dimension. The two-dimensional data were processed with Gaussian
apodization in both dimensions. The stoichiometry of the peptide with the oleic
acid was determined by comparing the peak areas (using the 1D 1H spectra) or
peak volumes (using the 2D 1H–13C HSQC spectra) of well-resolved, isolated
regions found in the spectra.
Diffusion-ordered spectroscopy (DOSY) measurements were performed at 293
K. Samples were prepared in 50 mM phosphate buffer at pH 7.4. The
DgscteSL_dpfgsc DOSY pulse program was used, which consists of gradient
compensated stimulated echo with spin lock using the excitation sculpting solvent
suppression method47. A spectral window of 13,020 Hz was used, with an
acquisition time of 2.46 s with a relaxation delay of 3 s. The FIDs were collected
with 32,000 complex data points with 64 scans. Logarithmically the gradient pulse
strength was increased from 3% to 86% of the maximum strength of 32,767 G/cm
in 60 steps. A diffusion time (Δ) of 100 ms and bipolar half-sine-shaped gradient
pulses (δ) of 5 ms was applied. 1,4-Dioxane, which is known to behave
independently of protein concentration and the folded state of the protein, was
used as an internal chemical shift reference and hydrodynamic radius calibration
reference (3.75 ppm; R
H
=2.12 Å)48,49. DOSY processing was performed using a
two-component fit with a discrete approach, which further processed using a non-
uniform gradients approach. Three replicate acquisitions were given for each
sample, and the resulting diffusion coefficient (D) values calculated. For alpha1
peptide the average Dvalue was 2.162 and 14.10 m2/s for 1,4-dioxane. In the case
of alpha1–oleate complex the average Dvalue was 0.986 m2/s for complex and
13.61 m2/s for 1.4-dioxane. The calculated R
H
are as follows: alpha1 peptide R
H
=
13.82 ± 0.447 Å, alpha1–oleate complex R
H
=29.3 ± 0.606 Å, HSA R
H
=40.9 ±
1.44 Å, oleate in aqueous solution R
H
=104.3 ± 7.22 Å, oleate in methan ol R
H
=
5.58 ± 0.0649 Å. Note that the D(diffusion coefficient) values for 1,4-dioxane are
slightly variable dependent upon the co-solute (lower panel where Dis between
14.4 and 12.6), which rightly reflects the different solution micro-environment
conditions that both solutes are mutually experiencing for each sample.
For T
2
relaxation measurements, the standard CPMGT2 pulse sequence was
used to run the experiments with 15 relaxations delays, which were chosen
logarithmically for different maximum T
2
time intervals: 8 s (alpha1 peptide), 1.2s
(alpha1–oleate complex), 3.0 s (HSA), 7.0 s (oleate in aqueous solution), and 10 s
(oleate in methanol), respectively. The data were acquired with 32,000 complex
points with a baseline correction of 4. The T
2
analyses were performed on VNMRJ
version 4.0 (Agilent Technologies) software by the exponential fitting of these
values with their corresponding intensity. All other NMR parameters were kept
constant for all samples throughout the experiments. The experiments were
acquired at a sample temperature of 293 K. The data are presented in
Supplementary Table 1.
Size-exclusion HPLC. Calibration standards and samples were injected onto a
TSKgel Super SW3000 HPLC column (4.6 mm × 30 cm, Particle size 4 μm, pore
size 25 nm, Tosoh Bioscience) eluted with 0.05 M sodium phosphate buffer pH 7.0
containing 0.1 M Na
2
SO
4
at a flow rate of 0.25 mL/min and detection at 280 nm.
The chromatography was performed on a Dionex Ultimate HPLC 3000 Standard
System running Chromeleon 6 software (Dionex, Thermo Scientific). The standard
calibration curve was generated with the proteins given including HSA, which has a
hydrodynamic radius (R
H
) of 40 Å (Supplementary Fig. 5). The R
H
vs. elution
volume linearity of the standard calibration curve is known to vanish after
approximately 3.8 mL of elution volume50. As a result, the R
H
of oleate in methanol
eluent (a solvent that ensures that the fatty acid is monomeric) will be less than
what is estimated from the standard curve (12.3 Å). The retention times of small
R
H
-analytes are closely reproduced regardless of eluent, be it aqueous buffer or
methanol.
Computational simulations: model building of peptide and peptide–OA complexes.
The initial structure of the alpha1 peptide was obtained from the corresponding
domain in the crystal structure of human alpha-lactalbumin (PDB ID: 1B9O). All
cysteines were mutated to alanines, consistent with findings that a reduced human
alpha-lactalbumin mutant in which all cysteines mutated to alanines could form a
cytotoxic complex in the presence of the lipid cofactor9. The initial structure of the
sar1alpha peptide was obtained from an I-TASSER-built homology model51. The
sequence similarity of the Sar1alpha peptide with the sequence of the top-ranked
threading template used by I-Tasser (PDB ID: 1R7G) was 0.35. However, this is
eventually irrelevant as we used extensive H-REMD sampling to sample the con-
formations of the peptide, both in its oleate bound and apo forms. The alpha1 and
the sar1alpha peptide were centered in a cubic box with box edges 1.2 nm from the
peptide. For the oleate-containing systems of alpha1 and sar1alpha, 4 molecules of
oleate are placed randomly in the box surrounding the alpha1 peptide to obtain a
peptide–oleic acid ratio of 1:4. The Amber 99SB-ildn52 force field and the TIP3P53
water model were used. For the coordinates, the starting structure was built using
Discovery Studio 4.1 (Accelrys). Geometry optimization for the ligand was per-
formed using Gaussian0954 at the level of HF-6-31G*, and the partial charges were
determined by the RESP55 method implemented in the antechamber tool of
AmberTools16 (AMBER 2016). Topologies for the oleate were built using the
General Amber Force Field56. The respective atom labels, corresponding atom type,
and partial charges are shown in Supplementary Table 10. Additional parameters
Fig. 7 Reprogramming of gene expression. RNA sequencing was used to compare gene expression profiles in tumor tissue biopsies from the treatment or
placebo groups. aPie chart of genes regulated in response to treatment (cut-off FC > 1.5, P< 0.05 compared to the placebo group). In the treatment group,
82% of all regulated genes were cancer-related and 14% were bladder cancer-related. Gene categories were identified by biofunction analysis. bHeatmap
of specific cancer- and bladder cancer-related genes regulated in tumor biopsies from the treatment group (red =upregulated, blue =downregulated, cut-
off FC > 1.5, P<0.05 compared to placebo group). About 60% of all regulated genes were inhibited in the treatment group. cDetailed analysis of data in (a,
b). Top regulated, cancer-associated functions are shown. Inhibition is indicated by negative z-scores (blue) and significance by Pvalues (orange). The
expression of genes involved in tumor invasion, neoplasia, tumor growth, and urinary tract tumors was strongly inhibited. dInhibition of Ras signaling in the
treatment group compared to placebo. eBladder cancer gene network regulated specifically in patients receiving alpha1–oleate treatment compared to
placebo.
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Content courtesy of Springer Nature, terms of use apply. Rights reserved

for the molecule are derived from the General Amber Force Field. For reference,
the structure of the oleate molecule and its atom labels is shown in Supplementary
Fig. 11. All systems were neutralized and Na+and Cl−ions were added to a
concentration of 0.15 M. Energy minimization was performed using the steepest
descent algorithm for 1000 steps to remove any initial bad contacts. Long-range
electrostatics were treated with the particle mesh Ewald algorithm57, with a real-
space cutoff of 1.2 nm, and Van der Waals interactions were truncated at 1.2 nm.
All systems with oleate-containing peptides or naked peptides were initially heated
at 500 K for 40 ns to eliminate starting structure bias and provide a partially
unfolded state for the peptides. Temperature coupling of the system was performed
using a velocity rescaling thermostat58.
Hamiltonian replica exchange molecular dynamics simulations. The Gromacs 5.1.2
molecular dynamics package56 with the Plumed 2.3 plugin for Hamiltonian Replica
Exchange Molecular Dynamics59 was used to perform the simulations. All atoms of
the alpha1- and sar1alpha peptide residues, along with the oleate residues of the
two oleate-containing systems, were selected for Hamiltonian scaling. Twenty
replicas were used for each system, and scaling factors were generated for an
effective temperature range of 300–800 K. Temperatures for scaling were selected
based on a geometric progression. The temperature factors were 300, 315.893,
332.629, 350.251, 368.807, 388.346, 408.919, 430.583, 453.395, 477.415, 502.707,
529.34, 557.384, 586.913, 618.006, 650.747, 685.223, 721.525, 759.75, and 800 K.
Each replica was simulated for 400 ns, resulting in an effective simulation of 8 µs.
Exchanges were attempted every 2 ps, and the result was an average acceptance
probability of approximately 30%.
Simulation analysis. Analysis of simulation data was performed using the built-in
Gromacs tools of the Gromacs package56. The ensemble for each system with the
canonical unscaled potential energy was used for the analysis, and data analysis was
performed on the last 300 ns for each system. Dihedral principal component
analysis60 was performed using the gmx angles, gmx covar, and gmx anaeig tools to
prepare and diagonalize the covariance matrix and analyze eigenvectors and
eigenvalues. The free-energy surface was constructed through projection onto the
first and second principal components with the formula F
i
=−RT ln(P
i
/P
o
), where
Ris the gas constant, Tis temperature (300 K), P
i
is the population in each bin and
P
o
is the population of the most populated bin. The gmx cluster tool of Gromacs
5.1.2 was used to identify the representative structure of each minima for geometric
clustering for the Gromos algorithm. We used the define secondary structure of
proteins (DSSP)61 algorithm to calculate secondary structure propensities. For our
analysis, we classified the 3
10
helix, αhelix, and πhelix structures as helices; the β-
sheet and residue in isolated β-bridge structures as sheets and the remaining
structures as others. The contact probability was calculated using the gmx mindist
tool in the Gromacs package. The minimum distance between protons of side
chains for each residue and oleic acid was calculated for each frame. To calculate
the contact probability, a contact was defined if the measured distance was less than
0.55 nm. The contact probabilities between Aromatic ring protons and Olefinic
protons of alpha1- and sar1alpha–oleate-containing systems were also calculated
similarly. Proton distances were calculated to facilitate the comparison of simu-
lation data to Nuclear Overhauser Spectroscopy data.
Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
Data availability
The data supporting the structural and cellular findings of this study are available within
the article and its supplementary information files. The structural data referenced during
the study are available in a public repository from the Protein Data Bank website (www.
rcsb.org, DOI:10.2210/pdb1B9O/pdb, DOI:10.2210/pdb1R7G/pdb). The RNA
sequencing data generated in this study have been deposited in the Gene Expression
Omnibus (GEO) database under accession number GSE172112. Source data are provided
with this paper.
Received: 8 April 2020; Accepted: 15 April 2021;
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Acknowledgements
The authors thank Petr Bouška and NEOX for the Monitoring of the clinical study;
Jeanette Valcich and the Center for Translational Genomics (CTG) at Lund University
for the mRNA Library prep and Sequencing; Susanne Strömblad, Lina Gefors and the
Lund University Bioimaging Centre (LBIC) for providing experimental resources for
tissue analysis, Arunima Chaudhuri for input and comments on the manuscript, Sara
Mangsbo, Uppsala University, Sweden for the MB49 cells (RRID:CVCL_7076). We
gratefully acknowledge the support of the Swedish Research Council, the Swedish Cancer
Society (Cancerfonden) and HAMLET Pharma. Support to the Svanborg group was
further provided from the European Union’s Horizon 2020 research and innovation
program under grant agreement No. 954360. The funding sources had no role in the
design of this study or in its execution, analyses, interpretation of the data, or decision to
submit the results for publication.
Author contributions
All authors met the ICMJE criteria for authorship. C.S., M.B., J. Ho, Y.G.M., K.H.M.,
conceived and designed the study. J. Ho, P.S.K., A.H., D.L.F., K.H.M. performed struc-
tural biology experiments and analyses. J.T.Y.-N. performed and analyzed the
simulations under Y.G.M. supervision. J. Ho, A.N., T. Hiep T., P.E. performed
cellular experiments and analyses. A.B., J. Háček, I.A., D.S.C.B., M.L.Y.W., T. Hiep T.,
H.N., J. Horňák, M.B., and C.S. performed the human trial and analyses. M.B., A.B.,
J. Háček, I.A., D.S.C.B., T. Hiep T., T. Hien T., M.L.Y.W., P.S., M.B., C.S. analyzed the
data and J. Ho, I.A., D.S.C.B., T. Hiep T., K.H.M., M.L.Y.W., A.B., M.B., C.S. wrote
the paper.
Funding
Open access funding provided by Lund University.
Competing interests
C.S. holds shares in HAMLET Pharma, as a representative of scientists in the HAMLET
group. Patents protecting the use of the alpha1 peptide were filed previously (Biologically
active complexes and therapeutic uses thereof; GB 201707715 priority date 14/05/2017,
PCT/EP2018/062396 filing date 14/05/2018; inventors: C.S., A.N., J.Ho). No specific
patents have been filed based on this study. Other authors declare no competing or
conflicts of interests.
Additional information
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41467-021-23748-y.
Correspondence and requests for materials should be addressed to C.S.
Peer review information Nature Communications thanks Keith Syson Chan and the
other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer
reviewer reports are available.
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