Novel Photoimmunoconluqates for Use in Photodynamic Therapy
Field of the invention
Photodynamic therapy (PDT) is a promising and minimally invasive approach for
the treatment of cancer. Following the introduction of improved photosensitizers
and clinical application protocols, several FDA-approved PDT drugs have become
available and others are in various stages of preclinical and clinical develop
ment 1. The photosensitizing agent can exert its effect when activated by nonhazardous
light directly, by becoming cytotoxic, or indirectly, by initiating the in
situ production of toxic free radicals or reactive oxygen species (ROS). These
processes cause damage to cells and ultimately induce cell death by apoptosis or
necrosis 2. The site of cellular damage depends on the photosensitizer type, the
incubation period and the mode of delivery. Hydrophobic photosensitizers tend to
damage cell membranes, whereas cationic photosensitizers accumulate within
membrane vesicles such as mitochondria and cause local damage 3.
One of the greatest challenges in PDT is the lack of targeting specificity. Photo¬
sensitizers damage healthy tissue as well as tumor tissue after activation by
light, and this can result in prolonged skin photosensitivity 4. To increase the
specificity of PDT, photosensitizers have been conjugated to tumor-specific monoclonal
antibodies or single chain antibody fragments (scFv), resulting in socalled
photoimmunoconjugates that deliver the photosensitizer directly to the
tumor tissue. This approach is known as photoimmunotherapy (PIT) 5. Standard
coupling reactions are unsuitable for the conjugation of photosensitizers and an¬
tibodies because there is no reliable way t o ensure that antibody-photosensitizer
conjugates are produced in the optimal stoichiometric ratio 6. Furthermore, the
chemical properties of the photosensitizer (e.g. hydrophobicity and the number
and arrangement of charged groups) can after the pharmacokinetic properties
and biodistribution of the antibody, finally causing non-specific binding and inter¬
nalization behavior. Random conjugation can also induce the self-quenching of
photosensitizer-excited states, thus reducing photodynamic activity 5. More con¬
trolled conjugation reactions are therefore required to overcome these limita¬
tions.
One of the main drawbacks of PDT is the non-selective effect of activated photosensitizers,
which tend to damage healthy as well as tumor cells. Targeted t her
apy using antibodies has revolutionized cancer treatment and several antibodies
that bind to tumor ceil antigens have achieved blockbuster status. The efficacy of
therapeutic antibodies can be improved by their covalent conjugation to addi¬
tional effector molecules (e.g. radio nuclides, drugs or toxins) 7, as this achieves
selective delivery and should reduce the systemic toxicity traditionally associated
with small molecule drugs 8. The same principle can be applied to photosensit iz
es. Effector molecules are generally conjugated to antibodies using either the
reduced sulfhydryl groups of cysteine residues or amino groups in lysine side
chains. However, both methods yield heterogeneous products, comprising a mix¬
ture of conjugated antibodies with the effector attached at different sites, and a
variable number of effectors attached to each antibody resulting in a range of
molar rations and very different pharmacokinetic, efficacy and safety profiles.
Hamblett and colleagues 9 have studied the toxicity, pharmacokinetic properties
and therapeutic efficacy of heterogeneous antibody-drug conjugates by purifying
three antibody fractions containing two, four and eight conjugated molecules of
monomethyl-auristatin E ( AE) . The fraction with eight MAE groups was
poorly tolerated and rapidly cleared compared to the other fractions, and demonstrated
the lowest efficacy. This suggests that the key design parameter for ant i
body-drug conjugates is the number of drug molecules attached to the antibody.
However, even purified antibodies carrying the same number of drug molecules
still constitute a complex mixture because of the many alternative attachment
sites. For example, there are approximately 40 lysine residues in a typical antibody,
potentially resulting in more than one million different conjugated antibody
species. Similarly, there are between one and eight cysteine residues, typically
generating approximately 100 different conjugated variants. Each version of the
antibody-drug conjugant typically displays a unique and unpredictable pharma¬
cokinetic profile 9.
Summary of the invention
Cancer cells can be killed by photosensitizing agents that induce toxic effects
when exposed to non-hazardous light, but this also causes significant damage to
surrounding healthy cells. The specificity of photodynamic therapy can be increased
by conjugating photosensitizing agents t o antibodies and antibody f rag
ments that bind specifically t o tumor-associated cell surface antigens. However,
standard conjugation reactions produce heterogeneous products whose targeting
specificity and spectroscopic properties can be compromised.
I n this invention, an antibody fragment (scFv-425) has been used that binds t o
the epidermal growth factor receptor (EGFR) as a model t o investigate the use of
SNAP-tag fusions as an improved conjugation strategy. The scFv-425-SNAP-tag
fusion protein allowed the specific conjugation of a photosensitizer, such as
chlorin e6, modified with 0(6)-benzylguanine, generating a homogeneous product
that was delivered specifically to EGFR+ cancer cells and resulted in signif i
cant, tumor cell-specific cytotoxicity. The impact of our results on the develop
ment of photodynamic therapy is discussed.
The present invention provides a compound comprising a photosensitizer covalently
coupled t o a binding structure selected from the group consisting of antibodies
or their derivatives or fragments thereof, synthetic peptides such as scFv,
mimotopes, which binding structure binds to CD antigens, cytokine receptors,
interleukin receptors, hormone receptors, growth factor receptors, more particu¬
larly tyrosine kinase growth factor receptor of the ErbB family, wherein the pho¬
tosensitizer is coupled t o the internalizing receptor binding protein via the modified
human DNA repair protein 06-a!kylguanine-DNA alky!transferase (hAGTm).
I n one embodiment of the present invention the epidermal growth factor recep¬
tor binding protein is a scFv antibody fragment, in particular the scFv antibody
fragment of the Seq ID No 1, encoded by the polynucleotide sequence of Seq ID
No 2.
I n another embodiment of the invention the compound of the invention compris¬
es or has the amino acid sequence Seq ID No 3 , encoded by the polynucleotide
sequence of Seq ID No 4.
In yet another embodiment of the compound of the invention the photosensitizer
is coupled at the active site of the 06-alkylguanine-DNA alkyltransferase.
In the compound of the invention the photosensitizer is selected from the group
consisting of porphyrins, chlorophylls and dyes having photosensitizing power.
Subject matter of the invention is also a compound devoid of a photosensitizer.
The compound comprises a binding protein selected from the group consisting of
antibodies or their derivatives or fragments thereof, synthetic peptides such as
scFv, mimotopes, which binding protein binds CD antigens, cytokine receptors,
interleukin receptors, hormone receptors, growth factor receptors, more particu¬
larly tyrosine kinase growth factor receptor of the ErbB family, which is cova!ently
coupled to a modified human DNA repair protein called 06-alkylguanine-DNA
alky!transferase (hAGTm)
In particular the binding protein is an scFv antibody fragment, in particular the
scFv antibody fragment of the Seq ID No 1 and/or Seq ID No 3. This compound
can be encoded by a polynucleotide having the sequence of Seq ID NO 2 and/or
Seq ID No 4. The specific embodiments bind t o tyrosine kinase growth factor re¬
ceptor of the ErbB family.
A specific embodiment of the compound is encoded by the polynucleotide of the
nucleotide sequence of Seq ID No 5.
Another subject matter of the invention is a method for manufacturing the com¬
pound of the invention comprising the step of fusing 06-alkylguanine-DNA
aIkytransferase (hAGTm) with a binding protein selected from the group consist¬
ing of antibodies or their derivatives or fragments thereof, synthetic peptides
such as scFv, mimotopes, which binding protein binds CD antigens, cytokine re¬
ceptors, interleukin receptors, hormone receptors, growth factor receptors, more
particularly tyrosine kinase growth factor receptor of the ErbB family. In part icu
lar, the scFv-425 DNA sequence is inserted into the Sifl and Notl-digested site of
eukaryotic expression vector p S SNAP providing an N-terminal binding ligand
(scFv-425) and a C-terminal SNAP-tag sequence.
I n particular, a His tag is also fused to the protein. The fused protein can be ex¬
pressed in human cells in particular in embryonic kidney cell line such as HEK-
293T cells (ATCC: CRL- 11268) and purified using an affinity resin for the tag for
example a Ni-NTA modified resin.
Also subject matter of the present invention is a porphyrin derivative of the for¬
mula
Chlorin e6 C H O
wherein the carboxyi groups of the porphyrin photosensitizer, such as chlorin e6,
are at least partially reacted to an activated ester or by a coupling agent, fol¬
lowed by reacting with 06-benzylguanine, 02-benzylcytosine or a coenzyme A
(CoA).
In the method of the invention 06-benzylguanine, 02-benzylcytosine or a coenzyme
A (CoA) a coupled to a linker molecule, such as PEG-24-NH 2 and/or the
activated ester is formed by succinimides, such as NHS, or the coupling agent
selected from the group consisting of a carbodiimide, such as EDC, EDAC and
DCC.
Subject matter of the invention is aiso a medicament comprising the compounds
of the invention and a pharmaceutically acceptable adjuvant for improving or
render possible the pharmaceutical effect associated with the
photoimmunotherapy.
The invention is also providing a use of the compounds of the invention for t reat
ing cancer by photoimmunotherapy.
The skilled person knows that the term "comprising" can be replaced by " consist
ing" without introducing new subject-matter extending beyond the matter dis
closed herein.
The effects of the compound of the invention are demonstrated and more de¬
tailed described in the following by means of specific examples. The epidermal
growth factor receptor (EGFR, erbBl, HER1), one of four member of the ErbB
family of tyrosine kinase growth factor receptors, is overexpressed in approximately
30% of epithelial cancers and has thus become an attractive target for
cancer immunotherapy 10 . The recombinant anti-EGFR antibody fragment scFv-
425 binds t o EGFR on the surface of cancer cells and induces receptor internali¬
zation efficiently 11. scFv-425 is used as a model for the development of a new
conjugation strategy t o improve the specificity and efficacy of PIT. To achieve
these aims the SNAP-tag technology has been used which is based on a 20-kD a
modified human DNA repair protein called 06-alkylguanine-DNA alkyltransferase
(hAGTm) that was initially developed for the site-specific labeling of antibodies
with opticaily-active moleculess 11 . The SNAP-tag allows efficient, covalent cou
pling to any substrate modified with the acceptor group 0(6)-benzylguanine
(BG). The SNAP-tag reacts with para-substituted BG derivatives by transferring
the substituted benzyl group t o its active site via a nucleophilic substitution reac¬
tion and releasing free guanine 1.
According t o the invention a scFv-425-SNAP-tag fusion protein was designed and
synthesised. A BG-modtfied chlorin e6 (Ce6) photosensitizer was delivered t o
EGFR+ cancer ceils. The construct also included a linker region and 24 polyeth¬
ylene glycol (PEG) chains to increase the distance between the photosensitizer
and the protein. The BG-modified Ce6 was conjugated specifically and covaiently
to the scFv-425-SNAP-tag fusion protein with no detrimental impact on the bind
ing and internalization activities of the antibody. Ce6 was delivered specifically to
four EGFR+ carcinoma cell lines (A431, MDA-MB-231, MDA-MB-468 and SiHa)
and resulted in significant, tumor cell-specific cytotoxicity.
Detailed description of the invention
The present invention is further exemplary described in greater detail using Ce6
as photosensitizer and tyrosine kinase growth factor receptor of the ErbB family
as the binding.
Legends of the Figures
Figure 1: Construction, expression and binding of the SNAP-tag fusion proteins
(a) Schematic diagram of the bictstronic eukaryotic expression cassettes for the
recombinant SNAP-tag fusion protein. The pMS-scFV-425-SNAP vector encodes
binding ligand (scFv-425) which is under the transcriptional control of the C V
promoter, and joined in-frame to the SNAP-tag. An immunoglobulin k leader se
quence (Ig- K -L) facilitates protein secretion, and a TGA stop codon is placed
immediately after the C-terminal HiS6-tag. The expression cassettes for the con
trol vector were the same as the PMS-scFv-425-SNAP but contain scFv-Ki4 instead
of scFv-425 as a binding ligand. ( b ) Purification fractions of scFv-425-SNAP
protein were separated by SDS-PAGE, and then stained with coomassie brilliant
blue, (c) scFv-425-SNAP was incubated with BG-Vista green, proteins were visu
alized with UV light. M: Protein marker, 1: 3m I of eluted scFv-425-SNAP with
250mM Imidazoi, 2 : 1.5 m I of eluted scFv-425-SNAP with 250mM Imidazol, 3 :
IOmI of eluted scFv-425-SNAP with 40mM Imidazol, 4 : eluted protein with l Om
Imidazol, 5 : Flow through, 6 : HEK-293T cells supernatant. Binding analysis of
scFv-425-SNAP and scFv-Ki4-SNAP were assessed by flow cytometry using
EGFR' A431 (d) and EGFF L540 cells (e) . Filled gray curves represent untreated
cells. Cells were incubated with 0.5 pg/ml of the purified fusion protein scFv-425-
SNAP (light gray curve) and Ki4-SNAP (black curve). As a secondary antibody a
Penta-His Alexa Fluor 488 Conjugate (dilution 1/500) (Qiagen) was used. To ex
clude nonspecific staining of the anti-His Alexa Fluor 488 detection antibody,
omission of the His-tagged fusion protein served as control (dotted black
curves).
Figure 2 : Analysis of Ce6 photosensitizer by mass spectrometry before and after
modification with benzylguanine (BG). (a) ESI mass spectrum of Ce6, BG-PEG24-
NH , and BG-PEG24-Ce6. The top panel represents Ce6 (597.215 Da), the middle
panel represents BG-PEG24-NH 2 (1398.761 Da) and the bottom panel represents
BG-PEG24-Ce6 (1979.004 Da) (b) Coupling of BG-PEG24-Ce6 to scFv-425-
SNAP. , protein marker; 1, scFv-425-SNAP incubated with a 1.5-fold molar ex
cess of BG-VistaGreen; 2, scFv-425-SNAP blocked with a 3-fold molar excess of
bromothenylpteridine (BTP), incubated with BG-Ce6 for 2 h, and finally mixed
with BG-VistaGreen; 3, scFv-425-SNAP incubated with a 1.5 -fold molar excess
of BG-Ce6 for 2 h, then a 1.5-fold molar excess of BG-VistaGreen. Coupled proteins
were separated by SDS-PAGE and visualized with the CRi Maestro Imaging
System, The different dye spectra were unmixed using Maestro software and the
corresponding gel was stained with Coomassie Brilliant Blue (c).
Figure 3 : The binding activities of scFv-425-SNAP-VistaGreen and scFv-425-
SNAP-Ce6, which specifically recognize EGFR+ cells. Flow cytometry analysis was
carried out after incubating 4 x 105 cells with each fusion protein for 20 min at
37°C in PBS. (a) The scFv-425-SNAP-VistaGreen (light gray curve) was tested
against A431, MDA-MB-468, MDA-MB-231, SiHa, L540, and CHO-K1 cells (filled
gray curve). As a control, scFv-Ki4-SNAP was labeled with BG-VistaGreen (black
curve) and its binding activity was tested against A431, L540 and CHO-K1 ceiis
(filled gray curves), (b) The binding efficiency of scFv-425-SNAP-Ce6 (light gray
curve) was tested against A431, MDA-MB-468, MDA-MB-231, SiHa, L540 and
CHO-K1 cells (filled gray curves). As a control, scFv-Ki4-SNAP labeled with BGCe6
(black curve) was tested against A431, L540 and CHO-K1 cells (filled gray
curves).
Figure 4 : internalization of fusion proteins analyzed by confocal microscopy.
Confoca! images were obtained for the EGFR+ cell lines A431, MDA-MB-468,
MDA-MB-231 and SiHa, and for the EGFR cell lines L540 and CHO-K1 incubated
with 0.5 pg scFv-425-SNAP-Ce6 for 30 min at 4°C (a) or for 60 min at 37°C (b).
(1) Ce6 fluorescence signal; (2) transmitted light; (3) overlay of fluorescence
signal and transmitted light.
Figure 5 : Evaluation of photodynamic therapeutic efficiency. Cell proliferation and
apoptosis assays were carried out using the scFv-425-SNAP-Ce6. The cytotoxici¬
t y of scFv-425-BG-Ce6 was determined against cell lines A431 ( ■) , MDA-MB-468
( A ) , MDA-MB-231 (¨), SiHa (·) and CHO-K1 (T) using the XTT assay on (a) ir¬
radiated cells and (b) non-irradiated cells. The cytotoxicity scFv-Ki4-SNAP-Ce6
against A431 cells ( ,) was tested as a control. The same cells were treated with
different concentrations of BG-Ce6 and cell viability was analyzed with (c) and
without (d) light activation, (e) Apoptosis was evaluated using the Apo-ONE™
Homogeneous Caspase-3/7 Assay, with 50 nM BG-Ce6, 200 nM scFv-SNAP-Ce6
and 200 nM scFv-Ki4-SNAP-Ce6. (f) The generation of reactive oxygen species
by illuminating photosensitized A431 cells, detected using the dichlorofluorescein
derivative carboxy-H2DCFDA.
Photodynamic therapy (PDT) is a minimally invasive treatment that uses non¬
toxic photosensitizers and harmless visible Sight in combination with oxygen to
produce cytotoxic reactive oxygen species that kill malignant cells by apoptosis
and/or necrosis 12 . Many different photosensitizers have been developed, but Ce6
has been chosen as a model because it has been evaluated extensively in PDT
studies and also has advantageous physical and chemical properties. Ce6 has an
absorption maximum at 664 nm, which is a good compromise between photon
efficacy and cell penetration 13, and the presence of carboxyl groups allows fur¬
ther functionaiization 5.
The use of SNAP-tag technology of the present invention provides a unique con¬
jugation site on the antibody, allowing the production of a homogeneous conju¬
gate preparation. The construct of the invention in which the coding sequence of
an scFv antibody that binds specifically t o EGFR was genetically fused t o the
hAGT cassette, endows the antibody with a SNAP-tag and therefore allows sitespecific
conjugation BG-modified substrates, in particular Ce6. This conjugation
method can be applied to any antibody-photosensitizer combination as long as
the antibody carries the SNAP-tag and the substrate is modified with a BG group.
The conjugation reaction was efficient, allowing the preparation of homogeneous
samples of scFv-425-SNAP-Ce6 and scFv-Ki4-SNAP-Ce6. These preparations
were tested for their ability t o kill tumor cells specifically. I t has been found that
scFv-425-SNAP-Ce6 selectively killed EGF + cells in four human tumor-derived
cell lines representing epidermal, breast and cervical carcinomas (A431, MDAMB-
231, MDA-MB468 and SiHa) after exposure to light. The phototoxicity of
scFv-425-SNAP-Ce6 was dependent on the presence of EGFR and light, and toxicity
was most potent in A431 and DA- B468 cells, which express the largest
amount of the receptor (1-1.3 x 106 receptors/cell) 14'15 . The other cells lines ex
pressed less EGFR (1.3xl0 5 receptors/cell for MDA-MB-231 and 2 x 10 - 2 x 105
receptors/cell for SiHa) 15,16 , and the toxicity of scFv-425-SNAP-Ce6 was concomi¬
tantly reduced, although not to the point where the fusion protein would be therapeuticaily
ineffective. This means that scFv-425-SNAP-Ce6 can target a wide
range of EGFR+ cells not only those with the highest expression levels. No toxici
ty was observed when EGFR cells (CHO-K1) were exposed t o scFv-425-SNAPCe6.
I t has been previously shown that scFv-425-SNAP accumulates directly in mouse
kidneys after injection, and is subsequently detected in the bladder, indicating
clearance by renal filtration 10 . Despite the rapid clearance, the accumulation and
retention of scFv-425-SNAP in tumor tissue was evidently sufficient t o yield very
high tumor t o background ratio 10 h post- injection.
Expression, purification and functional analysis of scFv/SNAP-tag fusion proteins
The coding sequences for the EGFR-specific scFv-425 antibody fragment 10 and a
control fragment (scFv-Ki4) 17 that binds to a different antigen (CD30) were
transferred to the pMS-SNAP bicistronic vector to generate the complete scFv-
425-SNAP and scFv-Ki4-SNAP cassettes, as shown in (Fig. la). The constructs
were introduced into HEK-293T cells by transfection and stably transformed cells
were identified by selection on zeocin and by monitoring green fluorescent pro¬
tein (GFP) activity. The fusion proteins were isolated from the culture superna¬
tant t o a final purity of ~90% by affinity chromatography (using the C-terminal
His tag) and the final yield was 18 mg/L of protein in the supernatant (Fig. lb).
The activity of the S AP tag was confirmed in each of the fusion proteins by mix¬
ing the unprocessed culture supernatant, the flow through fraction and the eluate
from the chromatography step with BG-modified Vista Green (Fig. lc). The bind¬
ing activity of the scFv-425-SNAP protein was confirmed by flow cytometry using
one target cell line expressing EGFR (A431), and one control cell line lacking this
antigen but expressing CD30 (L540). Binding was detected with a secondary anti-
His Alexa 488 antibody. Flow cytometry data confirmed the rapid and efficient
binding of scFv-425-SNAP specifically t o EGFR+ target cells (Fig. Id), whereas
scFv-Ki4/SNAP bound only t o the CD30+ L540 ceils (Fig. le).
Modification of the photosensitizer chlorin e6 with benzylguanine
The photosensitizer chlorin e6 (Ce6) was modified successfully using N-(3-
dtmethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), the sodium salt
of hydroxysulfosuccintmide (sulfo-NHS) and a BG-PEG24-NH2 linker. Ce6 carboxy
l groups were modified to BG groups, and the efficiency of the reaction was determined
by HPLC (data not shown). The high purity of BG-PEG24-Ce6 was con¬
firmed by mass spectrometry. The accurate masses of Ce6, BG-PEG24-NH2 and
BG-PEG24-Ce6 were detected on a Micromass QTOFII mass spectrometer, which
confirmed that purified BG-PEG24-Ce6 had the same mass as the theoretical
mass calculated for coupled Ce6 and BG-PEG24-NH2 (Fig. 2a)
Protein labeling with BG-modified f!uorophores and Ce6
The functionality of the SNAP-tag was tested by coupling t o BG-modified fluorescent
dye, which revealed a labeling efficiency of 85-90% after a 2-h incubation
at room temperature (data not shown). The reaction was repeated using BGmodified
Ce6. The photosensitizer reacted solely with the active SNAP-tag in the
fusion proteins and the reaction could be irreversibly blocked with the
bromothenylpteridine (BTP), as shown by post-incubation with a 1.5-fold molar
excess of BG-Vista Green. Analysis with the CRi Maestro imaging system showed
no fluorescence associated with the previously blocked fusion protein (Fig. 2b, c).
Flow cytometry and confocal microscopy
To determine the activity of labeled scFv-425-SIMAP fusion proteins, flow
cytometry analysis was carried out using proteins that had been labeled with either
BG-Vista Green or BG-Ce6. All the labeled proteins showed a strong f luores
cence signal on the corresponding target cell line (A431, MDA-MB-231, MDA-MB-
468 and SiHa) but not on control cells (L540 and CHO-K1) after a 30-min incuba¬
tion on ice. As expected, labeled scFv-Ki4-SNAP showed a strong fluorescence
signal on L540 but not on A431 and CHO-K1 cells (Fig. 3).
Confocal microscopy revealed strong, specific and homogeneous membrane
staining on A431, MDA-MB-231, MDA-MB468 and SiHa cells incubated with scFv-
425-SNAP-Ce6 (Fig. 4a). The labeled fusion protein was specifically and efficient¬
ly taken up into A431, MDA-MB-231, MDA-MB468 and SiHa cells after a 30-min
incubation at 37°C but not at 4°C (Fig. 4b). I n contrast, no signal was detected
when the EGFFT cell lines L540 and CHO-K1 were incubated with scFv-425-SNAPCe6
under the same conditions (Fig. 4a, b).
Photocytotoxicity of scFv-425-SNAP-Ce6
The concentration-dependent cytotoxic effects of scFv-425-SNAP-Ce6 and uncon¬
jugated BG-Ce6 were evaluated using an XTT-based co!orimetric cell proliferation
assay with the four EGFR+ cell lines and CHO-K1 as a negative controls. The v ia
bility of A431, MDA-MB-231, MDA-MB-468 and SiHa cells treated with scFv-425-
SNAP-Ce6 was reduced significantly, in a concentration-dependent manner, after
a 24-h incubation followed the light activation. The IC values were 48 n
(A431), 200 nM (MDA-MB-231), 38 nM (MDA-MB-468) and 218 nM (SiHa). CHOK
cells remained unaffected even when exposed to 800 nM of the conjugated
fusion proteins, and the control construct scFv-Ki4-SNAP-Ce6 had a negligible
effect in both A431 and CHO-K1 cells. In contrast, unconjugated Ce6 was toxic
towards all the cell lines, with IC values of 16 nM (A431), 22 nM (MDA-MB-
231), 22 nM (MDA-MB-468), 26 nM (SiHa) and 18 nM (CHO-K1). These data are
shown in (Fig. 5a,c).
Both the conjugated and unconjugated forms of Ce6 were toxic only after light
activation, as confirmed by carrying out parallel experiments without the light
activation step. No significant reduction in viability was observed in any of the
cell lines (Fig. 5b,d).
To determine whether scFv-425-SNAP-Ce6 selectively induced programmed cell
death in target cells by triggering the apoptotic pathway, the activity of caspase-
3 and caspase-7 has been analyzed in A431, MDA-MB-231, MDA-MB468, SiHa
and CHO-K1 cells 24 h after light activation. Both scFv-425-SNAP-Ce6 (200 nM)
and unconjugated Ce6 (50 nM) increased the levels of caspase-3 and caspase-7,
whereas no significant increase was observed in A431 cells treated with 200 nM
scFv-Ki4-SNAP-Ce6 (Fig. 5e).
The production of ROS in photoactivated A431 cells was investigated by measur¬
ing the 485/535-nm fluorescence of DCF, produced by the oxidation and
deacetylation of 6-carboxy-20,70-dichlorodihydrofluoresceindiacetatedi-(acetoxymethyl)
ester (H DCFDA). It has been found that a burst of ROS synthesis follows
light activation in the presence of 200 nM of the conjugated Ce6 and 50 nM of
the unconjugated Ce6, but there was only a small increase in ROS levels in nonirradiated
cells, barely above the background level observed in cells that were
not treated with the photosensitizer (Fig. 5f).
METHODS
Cell culture
All ceil lines were of human origin, including the EGFR+ A431, MDA-MB-231,
MDA-MB468 and SiHa cells, and the EGFR L540, CHO-K1 and HEK-293T cells.
A431, L540, CHO-K1 and HEK-293T cells were cultured in RPMI-I640 medium
supplemented with 2 mM L-glutamine, 10% (v/v) fetal bovine serum (FBS) and
100 U/mi penicillin-streptomycin. MDA-MB-231, MDA-MB468 and SiHa cells were
cultured in DMEM with 10% (v/v) fetal bovine serum (FBS) and 100 U/ml penicil¬
lin-streptomycin, All cells were incubated at 37°C in a 5% C0 atmosphere All
media and additives were obtained from Invitrogen, Darmstadt, Germany.
Protein expression and purification.
The sequence for each scFv was inserted into an expression cassette providing
an N-terminal binding ligand (scFv-425 or scFv-Ki4) and a C-terminai 06-
alkylguanine-DNA alkyltransferase (SNAP-tag) sequence. The TGA stop codon is
generated immediately after HiS6 tag sequence. His -tagged fusion proteins were
purified from cell-free supernatants by Ni-NTA metal affinity chromatography.
Larger volumes were purified on an Akta FLPC system with a 5-mL Ni-NTA
Superflow cartridge (Qiagen, Hilden, Germany) after equilibration with 4x buffer
(200 mM NaH P0 4, 1.2 M NaCl, 40 mM imidazole, pH 8). Bound His-tagged proteins
were eluted in 50 mM NaH P0 , 300 mM NaCl, 250 mM imidazole, pH 8).
After elution, proteins were dialyzed at 4°C overnight against phosphate-buffered
saline (PBS) containing 1 mM dithioerythritol (Carl Roth GmBH, Karlsruhe, Ger
many). Ectoine cryopreservative was added to a final concentration of 50 mM,
and aliquots were stored at -20°C.
Modification of Ce6 with benzylguanine
The carboxy! groups of Ce6 (Porphyrin Products, Logan, UT), were modified with
benzylguanine by mixing 2 mg Ce6 in dimethy!formamide for 30 min at room
temperature with a five-fold molar excess of EDC and sulfo-NHS (Sigma-Aldrich,
St Louis, MO). The activated mixture was then mixed with a four-fold molar excess
of the benzylguanine linker BG-PEG24-NH2 (Covalys Biosciences AG,
Witterswil, Switzerland) in the dark at room temperature overnight. The modified
Ce6 was purified by HPLC using a Shimadzu Prominence HPLC system, and a
2.5 m (4.6 x 50 mm) Water XBridge™ OSTC column (Waters, Milford, MA) at a
flow rate was 1 mL/min. Separations were carried out using a 20-min gradient
from 100% 0.1 M TEAA t o 100% acetonitrile, monitored at 280 and 410 nm. The
masses of Ce6, BG-PEG24-NH2 and BG-PEG24-Ce6 were confirmed using a
Micromass QTOFII mass spectrometer with an electrospray ion source Advion
Nanomate (Advion, Ithaca, NY, USA) 7m I sample volume, 1.4 kV. Accurate mass
es were derived from mass spectra in the range 300-2500 m/z using the
MaxEnt3™ algorithm (Micromass) in the range of 400-2000 Da.
Protein labeling
The purified SNAP-tag fusion proteins were conjugated with BG-modified dyes
(Covalys Biosciences AG, Witterswil, Switzerland) or BG-modified Ce6 by incuba¬
tion in the dark with a 1.5-3-fotd molar excess of dye for 2 h at room tempera
ture. Residual dye was removed by gel filtration chromatography using zeba spin
desalting columns, 7 K MWCO (Thermo Fisher Scientific, Rockford, IL). Coupling
efficiency was determined photometrically using the extinction coefficients of the
corresponding dyes and the theoretical extinction coefficient of the fusion pro¬
teins. Labeled proteins were visuaiized after separation by SDS-PAGE with either
a UV transilluminator Gel Doc XR gel documentation (Bio-Rad Laboratories,
Munchen, Germany) or a CRi Maestro imaging system (CRi, Woburn, MA, USA)
using the blue and yellow filter sets.
Flow cytometry
The binding efficiency of the labeled and unlabeled fusion proteins was deter¬
mined by flow cytometry using a FACSCalibur (Becton & Dickinson, Heidelberg,
Germany) and CellQuest software. EGFR+ cell lines A431, MDA-MB-231, MDAMB468
and SiHa were used to test the binding efficiency of scFv-425-SNAP, and
EGFR ceil lines L540 and CHO-K1 were used as negative controls. The control
fusion protein scFv-Ki4-SNAP recognizes the antigen CD30 and should therefore
bind t o L540 cells but not to the other cell lines. Approximately 4 l 05 cells were
incubated in 200 L PBS containing 0.5 g of labeled protein for 20 min on ice.
The cells were then washed twice with 1.8 mL PBS in a conventional cell washer
and analyzed by flow cytometry.
Confocal microscopy
Images were visualized with a TCS SP5 confocal microscope (LEICA Microsystem,
Wetziar, Germany). Cells were prepared as described above for flow cytometry.
Binding efficiency was determined by incubating cells with the labeled fusion proteins
for 30 in on ice. Internalization was monitored by incubating cells with
the labeled fusion proteins for 30 min at 37°C.
Phototoxicity of scFv-425-SNAP-Ce6
Aliquots of A431, MDA-MB-231, MDA-MB468, SiHa and CHO-K1 cells (2xl0 4) cul¬
tured as described above were washed twice in PBS and then treated with increasing
concentrations of either Ce6, scFv-425-SNAP-Ce6 or Ki4-scFv/SNAP-Ce6
followed by incubation for 3 h at 37°C. Control cultures were incubated with 500
g/ ml zeocin instead of the photosensitizer. The cells were then irradiated with
24 J/cm 2 broadband visible/near infrared light using Hydrosun type 505, 7-mm
water cuvette and orange filter OG590, spectrum in the range 580-1400 nm
(Hydrosun Medizintechnik GmbH, Mullheim, Germany) and incubated for a fur¬
ther 24 h at 37°C in a 5% C0 atmosphere.
Cell viability was determined using the XT cell proliferation kit I I (Roche, Mann¬
heim Germany), 24 h after light activation. Cells were incubated with 2,3-bis(2-
methoxy-4-nitro-5sulphonyl)-5[(phenyl-amino)carbonyl]-2H-tetrazolium hydroxide
reagent (Img/m!), and incubated for 2 h at 37°C. Reduction of XTT to
formazan by viable tumor ceils was monitored colorimetrically at an absorbance
wavelength of 450 nm and a reference wavelength of 630 nm using an ELISA
plate reader Elisareader ELx808 (Bio-TEK, Bad Friedrichsahll, Germany).
Caspase-3/7 activity in ce l iysates was determined using the Apo-ONE Caspase-
3/7 assay (Promega, Mannheim, Germany) 24 h after light activation. Briefly,
100 m I of Apo-ONE reagent was added to the cells, and they were incubated for 6
h before fluorescence readings were taken with an ELISA plate reader Elisareader
ELx808 (Bio-TEK, Bad Friedrichsahli, Germany) using an excitation wavelength of
485 nm and an emission wavelength of 535 nm. The concentration of ROS was
determined by measuring the 485/535 nm fluorescence ratio of H2DCFDA (Invitrogen,
Darmstadt, Germany). Briefly, 2xl0 4 cells were incubated in the pres
ence of 50 nM Ce6 or 200 nM scFv-425-SNAP-Ce6 and 10 mM H2DCFDA for 30
min in PBS containing 1% FCS. The cells were washed twice with warm PBS con
taining 2.5% FCS, cultured for 2 h in RPMI-160 medium and illuminated as de
scribed above. Fluorescence readings were taken directly after illumination. A
blank probe (cells and medium) reading was used as the background and subtracted
from all the sample readings.
Data Analysis
Statistical analysis and curve fitting were performed with GraphPad Prism soft
ware (GraphPad, San Siego, CA). Data are presented as the mean ± MES. St u
dent's t test and two-way analysis of variance were used t o assess the significance
of independent experiments. The criterion p < 0.05 was used to determine
statistical significance.
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Claims
1. A compound comprising
• a photosensitizer covalently coupled to
• a protein selected from the group consisting of
antibodies or their derivatives or fragments thereof, synthetic pep¬
tides such as scFv, mimotopes
• which bind CD antigens, cytokine receptors, interieukin receptors,
hormone receptors, growth factor receptors, more particularly tyro
sine kinase growth factor receptor of the ErbB family, wherein
• the photosensitizer is coupled to the binding protein via
06-a!kylguanine-DNA alky!transferase (hAGTm), a modified human
DNA repair protein.
2. The compound of claim 1, specifically targeting an internalizing and dis¬
ease-specific ceil surface receptor.
3. The compound of claim 1 or 2, wherein the tyrosine kinase growth factor
receptor binding protein is an scFv antibody fragment, in particular the
scFv antibody fragment of the Seq ID No 1, encoded by the polynucleo
tide sequence of Seq ID NO 2.
4. The compound of claim 1 to 3 with the amino acid sequence Seq I D No 3,
encoded by the polynucleotide sequence of Seq ID No 4.
5. The compound of claim 1 to 4, wherein the photosensitizer is coupled at
the active site of the 06-alkylguanine-DNA alkyltransferase.
6. The compound of claim 1 to 4 wherein the photosensitizer is selected
from the group consisting of porphyrins, chlorophylls and dyes.
7. A compound comprising
a binding protein selected from the group consisting of antibodies or their
derivatives or fragments thereof, synthetic peptides such as scFv,
mimotopes, which binding protein binds CD antigens, cytokine receptors,
interieukin receptors, hormone receptors, growth factor receptors, more
particularly tyrosine kinase growth factor receptor of the ErbB family,
which is covalently coupled to a modified human DNA repair protein
called 06-alkylguanine-DNA alkyltransferase (hAGTm).
8. The compound of claim 7 wherein the binding protein is a scFv antibody
fragment, in particular the scFv antibody fragment of the Seq ID No 1
and/or Seq ID No 3.
9. A polynucleotide sequence encoding the compound of claim 7 in particu¬
lar comprising Seq ID NO 2 and/or Seq ID No 4.
10. The polynucleotide of the nucleotide sequence of Seq ID No 5 encoding
the compound of claim 1.
11. A method for manufacturing the compound of claim 7 comprising the
step of fusing 06-alkylguanine-DNA alkyltransferase (hAGTm) with a
binding protein selected from the group consisting of antibodies or their
derivatives or fragments thereof, synthetic peptides such as scFv,
mimotopes, which binding protein binds CD antigens, cytokine receptors,
interleukin receptors, hormone receptors, growth factor receptors, more
particularly tyrosine kinase growth factor receptor of the ErbB family.
12. The method of claim 11 wherein the scFv-425 DNA sequence is inserted
into the Sifl and Notl-digested site of eukaryotic expression vector p SSNAP
providing an N-terminal binding ligand (scFv-425) and a C-terminal
SNAP-tag sequence.
13. The method of claim 11 or 12 wherein the scFv-425-SNAP fusion protein
is expressed in human embryonic kidney cell line in particular HEK-293T
cells (ATCC: CRL- 11268).
14. The method of any one of the claims 11 to 13 wherein the scFv-425-
SNAP fusion protein is purified from cell-free supernatant by an affinity
chromatograpy, in particular a Ni-NTA affinity chromatography.
15. A porphyrin derivative of the formula
coupled to the compound of claim 7.
16. A method of manufacturing of the compound of claim 15, wherein the
carboxyl groups of the porphyrin photosensitizer, such as chlorin e6, are
at least partially reacted to an activated ester or by a coupling agent, fol
lowed by reacting with 06-benzylguanine, 02-benzylcytosine or a coen
zyme A (CoA).
17. The method of claim 16, wherein 06-benzylguanine, 02-benzyicytosine
or a coenzyme A (CoA) a coupled t o a linker molecule, such as PEG-24-
NH and/or the activated ester is formed by succinimides, such as NHS,
or the coupling agent selected from the group consisting of a
carbodiimide, such as EDC, EDAC and DCC.
18. Medicament comprising the compound of at least one of the claims 1 to 8
and a pharmaceutically acceptable adjuvant for improving or render pos¬
sible the pharmaceutical effect associated with the photoimmunotherapy.
19. Use of a compound of at least one of the claims 1 t o 8 for treating cancer
by photoimmunotherapy.