ANN-CHARLOTT STEFFEN
Cellular retention of
125 I-labeled EGF-dextran
Master’s degree project
UPTEC X 01 011
Date of issue 2001-02Author
Ann-Charlott Steffen
Title (English)
Cellular retention of
125I-labeled EGF-dextran
Title (Swedish) Abstract
A tumor cell specific targeting molecule was constructed as a conjugate between epidermal growth factor, EGF, and dextran. EGF is the natural ligand of the EGF receptor that is over- expressed in some cancers and acts as the targeting part of the conjugate. Dextran is a glucose polymer that is not degraded by mammalian cells and can thus act as an effective carrier molecule, carrying radioactive nuclides for killing the tumor cell. The conjugate was tested for binding kinetics, specificity, internalization, retention and recharging. It was found that the binding was specific, most radioactivity was internalized and 20% of the initial radioactivity was left after five days when ~60% of the radioactivity was located on the dextran part of the
conjugate.
Keywords
EGF, dextran, tumor targeting, residualizing label, retention Supervisors
Åsa Liljegren
Division of Biomedical Radiation Sciences, Uppsala University Examiner
Jörgen Carlsson
Division of Biomedical Radiation Sciences, Uppsala University
Project name Sponsors
Language
English
Security
ISSN 1401-2138
Classification
Supplementary bibliographical information Pages
23
Biology Education Centre Biomedical Center Husargatan 3 Uppsala Box 592 S-75124 Uppsala Tel +46 (0)18 4710000 Fax +46 (0)18 555217
Ann-Charlott Steffen
Sammanfattning
De vanligaste behandlingsformerna mot cancer idag inkluderar kirurgi, cellgifter och extern strålbehandling. Kirurgi kan effektivt ta bort en stor tumör om den ligger bra till i kroppen, men det är svårt att få bort alla spår efter tumören, vilket ofta leder till att patienten återinsjuknar efter en tid. Därför används cellgifter som komplement till kirurgi för att försöka nå de spridda cancercellerna. Både cellgifter och extern strålbehandling har den egenskapen att de attackerar alla delande celler, alltså även friska celler. Detta leder till allvarliga sidoeffekter, vilket begränsar den dos man kan ge till patienten och det i sin tur leder till att en effektiv behandling av tumören kan bli omöjlig. Om man kunde målsöka enstaka tumörceller och attackera bara dessa skulle cancern kunna bekämpas effektivt utan att patienten utsattes för alltför mycket obehag.
I det här examensarbetet har en tumörspecifik målsökare undersökts med avseende på hur länge det går att få målsökaren att stanna i cancercellen, om man med hjälp av denna målsökare kan ackumulera radioaktivitet i cellen och om den är lika effektiv mot tillväxande och vilande celler.
Examensarbete 20 p i Molekylär bioteknikprogrammet Uppsala universitet Februari 2001
INTRODUCTION ... 2
MATERIALS AND METHODS... 6
CONJUGATION... 6
Radiolabeling ... 6
Reductive amination... 6
Gel filtration ... 6
Preparative Gel Electrophoresis ... 6
CDAP ... 7
Proteinase K degradation ... 7
CELLULAR TESTS... 8
Cell culture ... 8
Cell proliferation test ... 8
Conjugate binding test... 8
Displacement test ... 9
Retention – internalized / membrane bound ... 9
Long time retention (proliferating / non-proliferating cells)...10
Recharging (proliferating / non-proliferating cells)...10
RESULTS AND DISCUSSION ...11
CONJUGATION...11
Preparative Gel Electrophoresis ...11
Proteinase K degradation ...12
CELLULAR TESTS...12
Cell proliferation test ...12
Conjugate binding test...13
Displacement...14
Retention – internalized / membrane bound ...14
Long time retention...16
Recharging (proliferating / non-proliferating cells)...18
CONCLUSIONS ...20
ACKNOWLEDGMENTS...21
REFERENCES ...22
Introduction
Cancer has been a known disease for as long as we have a record of human history. In Sweden, one of three people will be diagnosed with cancer and cancer is responsible for one of five deaths in the country [10]. For many centuries surgery was the only treatment available, but this method was not always very effective due to difficulties removing the entire tumor. In the middle of the 19:th century it was discovered that tumors were caused by rapidly growing cancer cells and attempts to cure the patients by the use of poisons, such as arsenic, were made. This approach was however not as successful in curing the patients as it was at killing them. The discovery of anesthetics shortly afterwards, however, made more advanced forms of surgery possible and remained the most common cancer treatment. Over the last 60 years new methods have been developed to complement or substitute surgery [9].
The methods routinely used today - besides surgery - includes chemotherapy, hormone treatment and radiation therapy. The problem with surgery is, as discussed above, to remove the whole tumor and becomes impossible when metastases are present. Chemotherapy effects all dividing cells – not just tumor cells – and the side effects are often quite severe. Attacking the hormone system – as is done during hormone treatment – obviously causes unwanted side effects and this method is only possible on quite few types of cancer. Radiation therapy effects all cells or at least all cells within a part of the body. This limits the deliverable dose and thus the possibilities to cure the cancer [10].
An obvious way to diminish or avoid side effects would be to attack the tumor cells specifically with a toxin or radioactivity. To do that, a structure solely present on tumor cells or at least to a much higher degree must be found. The ideal tumor- seeking agent would attack a structure found only on tumor cells. It should also be non-immunogenic, stable in vivo, have good penetration characteristics and preferably be internalized by the tumor cells. Unfortunately no such molecule has yet been found. Monoclonal antibodies directed against structures found on tumor cells have been developed and are still the most common targeting molecules. They are, however, often not very tumor specific and they are also unstable in vivo and its size (~150 kDa) can effect penetration of the tumor. The latter problem is diminished by the use of antibody fragments, but the fragments often have a lower specificity than the intact antibody [21].
Some tumors over-express receptors for growth factors. One such receptor is the receptor for the epidermal growth factor, EGF. The receptors could be targeted with antibodies, but also with their natural ligands, that are much smaller. The disadvantage of using growth factors as targeting molecules is that in vivo degradation of the targeting agent occurs quite rapidly so the toxic substance is released from the
cell too soon. Previous experiments have shown that 90% of the bound radioactivity was lost, probably due to degradation, within two hours after incubation with EGF [3]. This problem can be solved however by covalently linking the growth factor to a carrier molecule that is not degraded and that can carry the toxic load necessary to kill the tumor cell, see Figure 1.
In this study EGF was used as targeting molecule and an 11-kDa dextran chain as a carrier – a residualizing label – labeled with 125I as “toxic load” since our only aim with the radionuclide was to trace the conjugate in the cells. For therapeutical purposes a more powerful nuclide, such as the -emitting 211At, can be used.
EGF has the advantage of being very small. Only 53 amino acids and 6 kDa should result in excellent penetration possibilities in tumor tissue. The EGF receptor has been found over-expressed in many different kinds of cancers, such as gliomas, bladder cancers and various types of squamous epithelial carcinomas and adenocarcinomas [1, 2, 5, 6, 11, 15, 20]. Numbers of 106 EGF receptors per cell have been reported [18]
whereas most normal cells have negligible amounts expressed (except for hepatocytes where 105-106 receptors per cell have been reported). This makes the EGF receptor a possible target for cancer therapy.
Dextran is a well-known blood volume expander used since the 1940’s. It is assumed to be non- immunogenic in most cases and is stable in vivo [21]. Dextran has also the
Figure 1 Targeting principle. Schematic representation of the tumor targeting principle. A targeting molecule attached to a carrier molecule with a toxic load binds to a receptor on the tumor cell membrane and is internalized in a vesicle. Proteolytic degradation of the targeting molecule occurs, and degradation products are excreted from the cell, while the carrier molecule remains internalized.
Targeting
molecule Carrier
Toxic load Receptor
Degraded growth factor
Endosome Lysosome
Cell membrane
advantage of being easily chemically modified to allow conjugation to other molecules.
In this study we used an established conjugation technique with reductive amination by the use of NaCNBH3. To get a homogenous conjugate murine EGF (mEGF) was used instead of human EGF (hEGF). mEGF has no lysines, so the only amino group available for the reductive amination reaction is the N-terminus while hEGF has two lysines, which gives three amino groups. Affinity towards the human EGF receptor is about the same for both types of EGF [21]. Tyrosine was coupled to the dextran part of the conjugate using the cyanylating agent CDAP. The chloramine-T method [8]
was then used to label tyrosines on both EGF and dextran with 125I. The obtained conjugate was then tested for its receptor binding capacity in vitro on cultured human glioma cells, over-expressing the EGF receptor.
The aims of this study were:
To investigate the long time retention of radioactivity for up to five days. Before this study, analysis of cellular retention studies had only been conducted up to 24 hours. A long retention is important for the possibilities to deliver a high dose - the longer the conjugate can remain intracellular the longer the radionuclide can irradiate the nucleus and the higher the probability for killing the cell.
To investigate whether the radioactivity dose after applying conjugate to its maximum level can be boosted to higher levels by applying a second dose, see figure 2. This is important for the same reasons as described above, for maximizing the dose delivered to each cell.
Figure 2 Schematic representation of an ideal boosting experiment. During phase 1 the first dose of the conjugate is applied and accumulated in the cells. The curve is flattened due to receptor saturation. During phase 2 the administration of conjugate is ceased and the cell-associated radioactivity is slowly decreasing. The third phase represents the second dose of conjugate, which leads to a higher level of cell-associated radioactivity due to receptor recruitment. Phase 4 represents the same phase as phase 2 but after the second dose of conjugate. The red dashed line represents what would happen if no second dose was administrated.
(1)
(3)
(2)
Time Cell-associated
radioactivity
(4)
To compare the effect of the conjugate on proliferating and non-proliferating cells in order to gain knowledge of the effect of the conjugate also on non-proliferating cells, that is found in certain areas in a tumor, see figure 3. Another reason for
performing experiments on non-proliferating cells is to facilitate calculations of the cell-associated radioactivity.
In addition to these three major aims, molecular weight distribution of molecules excreted from the cells after exposure to conjugate was to be briefly examined to look for differences between proliferating and non-proliferating cells. It should also be investigated whether the hypothesis that EGF degradation products are excreted after about one hour after conjugate binding as has been described for free EGF [4, 16] was correct also for EGF-dextran.
Necrotic region Non-proliferating region
Proliferating region
Figure 3 Schematic representation of a micro-metastasis showing the different regions that can be found.
Materials and methods
Conjugation Radiolabeling
mEGF (Chemicon, USA) was labeled with 125I (Amersham, Pharmacia Biotech, Sweden) in order to trace the EGF through the conjugation and purification procedures. The conjugate was once again labeled before cell tests were conducted.
The labeling method used was the chloramine-T method [8]. Na-125I (10-20 MBq) was added to a tube with 25 µl mEGF (0.1 mg/ml) or 25-50 µl EGF-dextran-Tyr conjugate (~1 µg). Addition of 10 µl (2 mg/ml, Sigma, USA, USA) chloramine-T started the reaction and after one minute of mixing, 25 µl (2 mg/ml, Aldrich, USA) sodium metabisulfite was added to stop the reaction. The labeled compounds (125I- EGF or 125I-EGF-dextran-Tyr-I125 conjugate) were then separated from excessive 125I using a NAP-5 column (Sephadex G-25, Amersham Pharmacia Biotech, Sweden) equilibrated with sodium phosphate buffer (pH 8.0) in the case of mEGF radiolabeling and PBS (Phosphate Buffer Saline) in the case of the conjugate. PBS was also used for diluting chloramine-T and sodium metabisulfite.
Reductive amination
About 70 mg dextran (11 0.5 kDa, purified from dextran T-10, Pharmacia Biotech, Sweden) was added to a tube with 24 mg NaCNBH3 (Merck, Germany), 100 µl mEGF (1 µg/µl) and 200 µl 125I-EGF (~1 µg EGF). The tube was kept under continuos stirring at room temperature for five days. The reaction is schematically described below.
Gel filtration
When the reductive amination reaction was finished, the conjugate was separated from low molecular weight compounds, such as free 125I and NaCNBH3. This was done using microspin G-25 columns (Pharmacia) equilibrated with 80 mM Tris-HCl with 0.02% bromophenol blue. The obtained fractions were measured with a NaI scintillation meter (Mini-Instruments LTD, UK).
Preparative Gel Electrophoresis
The conjugate was purified from free dextran and unbound EGF using a preparative gel electrophoresis (Mini Prep Cell, Bio Rad, USA) system that separates molecules according to charge. A 15 mm stacking gel (4%, Acrylamide:N.N Methylen- bisacrylamid 29:1, 125 mM Tris-HCl pH 6.8) was polymerized on top of a 45 mm separation gel (7%, Acrylamide:N.N Methylenbisacrylamid 29:1, 375 mM Tris-HCl
Dextran-CHO + mEGF Dextran-HC=N-mEGF NaCNBH3 Dextan-CH2-NH-mEGF
pH 8.8). The running buffer used was a Tris-Glycine buffer (25 mM Tris, 200 mM Glycine, pH 8.3) and the elution buffer was running buffer diluted ten times. A volume of 100 µl glycerol was added to the sample before application on the gel. The buffer flow was set to 50 µl/min and the electrophoresis was run at 300 V, 1 W, 3 mA (1W was kept constant, while other parameters were allowed to vary) for about 20 h.
Fractions of 1 ml were collected and analyzed using a gamma counter (1480 Wizard, Wallac, Finland). Fractions of 1 ml were collected and analyzed with a gamma counter and fractions containing the conjugate were pooled and freeze-dried to decrease sample volume and make a change of buffers possible. The sample was then dissolved in 500 µl water and desalted using a NAP-5 column equilibrated with water.
Fractions of 200 µl were collected and analyzed with a NaI scintillation meter and the conjugate fractions were pooled and freeze-dried again. Other peaks from the electrophoresis were further analyzed by gel filtration on a Superdex 75 column (Pharmacia, Sweden) equilibrated with PBS at a flow rate of 1 ml/min.
CDAP
The freeze-dried pool was dissolved in 25 µl water and put on ice with magnetic stirring. A volume of 25 µl (20 mg/ml) CDAP (1-cyano-4-dimethylamino pyridinium tetra-fluoroborate, Sigma, USA, USA) was added to the tube and the reaction taking place is described below as step 1. The mixture was kept on continuos stirring for 10 seconds before 10 µl TEA (Triethylamine, Sigma, USA) was added. After two minutes of stirring on ice 140 µl saturated tyrosine solution (Sigma, USA) was added and the reaction (described below as step 2) was carried out in room temperature for 18 hours.
The EGF-dextran-tyrosine conjugate was then separated from reagents and excess amounts of tyrosine by gel filtration on a NAP-5 column equilibrated with PBS.
Fractions of 200 µl were collected and analyzed with a NaI scintillation meter.
Conjugate fractions were pooled and stored at -20 C.
Proteinase K degradation
To analyze the quantity of 125I attached to the dextran part of the conjugate (this part is thought to remain intracellular for a long time) the conjugate was treated with proteinase K. Proteinase K degrades proteins by cleaving certain peptide bonds. The mEGF part of the conjugate is thus degraded, while the dextran part remains intact.
Two equal fractions of 25 µl (~10 kBq) freshly radiolabeled conjugate were treated with 42 µl proteinase K (25 U in 0.01 mM Tris-HCl buffer, pH 7.5, Roche) or water respectively, and were incubated at 42 C over night. The samples were separated on a
mEGF-dextran mEGF-dextran-
cyanate ester
mEGF-dextran-tyrosine Tyrosine
CDAP
(1)
200 400 600 800 1000 1200 1400
Molecular weight distibution
HMW LMW
(2)
200 400 600 800 1000 1200 1400
Molecular weight distibution
HMW LMW
Superdex 75 column equilibrated with PBS. The flow rate used was 1 ml/min and fractions of 1 ml were collected and analyzed for radioactivity content.
Cellular tests Cell culture
Human glioma U-343MGaCl2:6 with 8.6x105 EGF receptors per cell [19] were cultured as monolayers in Ham’s F-10 medium supplemented with 10% FBS (Fetal Bovine Serum, Biochrom, Germany), L-glutamine (2 mM, Biochrom, Germany) and PEST (penicillin 100 IU/ml and streptomycin 100 µg/ml, Biochrom, Germany) in an incubator at 37 C in humidified atmosphere equilibrated with 5% CO2. Medium was changed every two or three days and the cells were normally subcultured once a week. The cells were never trypsinated less then two days before experiments, since this treatment might damage the EGF receptors.
Cell proliferation test
Cell proliferation rate at different concentrations of FBS in the culture medium was measured in order to find a suitable concentration where the cells were neither increasing nor decreasing in number. The concentrations tested were 0%, 0.02%, 0.05% and as a control 10%. About 2x104 cells per dish were seeded and cultured in 35 mm culture dishes for one week. Medium was changed every two days. Once per day the cells in three dishes were washed once in ice-cold, serum free medium.
Trypsin-EDTA (0.25% Trypsin, 0.02% EDTA, Biochrom, Germany) was added (0.5 ml per dish) and the trypsination (in which the cells detach from the bottom of the dishes) was allowed to proceed for 10-15 minutes at 37 C. Addition of 1 ml complemented medium stopped the reaction. The cells were carefully resuspended and 0.5 ml of the cell suspension was used for cell counting in an electronic cell counter (Coulter Z2).
Conjugate binding test
EGF-dextran-tyrosine conjugate was radiolabeled, as described above, and diluted in complemented medium to an radioactivity concentration of about 38 kBq/ml. Culture dishes with ~3x105 cells each were washed once with ice-cold serum free medium and 1 ml of the labeled conjugate was added to each dish. The cells were incubated for different times, and triplicate dishes were analyzed. The dishes to be analyzed were washed six times with ice-cold serum-free medium, in order to remove all unbound compounds and then trypsinated with 0.5 ml trypsin-EDTA for 10-15 minutes at 37 C. Complemented medium to a volume of 1 ml was added and the cells were carefully resuspended. A volume of 0.5 ml of the cell suspension was used for cell counting and 0.5 ml for radioactivity measurement in a gamma counter.
Displacement test
A 1:10 dilution series of non-radioactive mEGF in complemented medium were made from 1000 ng/ml to 0.001 ng/ml. All culture dishes (~3x105 cells per dish) were washed once with ice-cold serum free medium and 0.5 ml of the different mEGF solutions and 0.5 ml conjugate solution, diluted in complemented medium to a radioactivity concentration of about 34 kBq/ml, were added to each dish. Triplicates of dishes exposed to each concentration were made. After 4 h of incubation at 37 C all dishes were washed six times with ice-cold serum free medium to remove unbound radioactivity. The cells were trypsinated as above, and cells were counted and radioactivity measured to determine the cell-associated radioactivity.
Retention – internalized / membrane bound
Culture dishes with ~1.5x105 cells were washed once with ice-cold serum free medium and 1 ml conjugate solution (radioactivity concentration about 37 kBq/ml) was added to each dish. The incubation at 37 C was terminated after 1 or 24 h and the cells were washed six times in ice-cold serum free medium. Complemented medium to a volume of 1 ml was added to each culture dish and the cells were incubated at 37 C again. After different times (0 to 25 hours) five dishes from each pre-incubation time were investigated. Two of them were washed and trypsinated, as previously described, and counted to get an average cell number. The other three dishes were used to determine the membrane bound and internalized radioactivity according to the method described by Haigler et al [7]. They were carefully washed and treated with 0.5 ml glycine-HCl (0.1 M, pH 2.5) for six minutes at 4 C. This treatment removes membrane bound radioactivity from the cells. The cells were washed with an additional 0.5 ml glycine-HCl and all glycine was collected for radioactivity measurement. The remaining radioactivity was assumed to be internalized, and was removed with 0.5 ml NaOH (1 M) at 37 C for one hour. NaOH was collected and analyzed in a gamma counter together with an additional 0.5 ml NaOH used for rinsing the dishes.
The distribution of high molecular weight compounds (HMW) and low molecular weight compounds (LMW) excreted from the cells during the retention phase were analyzed by separation on NAP-5 columns equilibrated with complemented medium.
A volume of 0.5 ml of the retention medium after 25 hours of retention was applied on the column and HMW compounds were eluted with 1 ml complemented medium.
An additional 1.5 ml eluted LMW compounds. The amount of radioactivity in the fractions was measured using a gamma counter. This analysis was done on retention medium both from dishes pre-incubated 1 h and 24 h with conjugate.
Long time retention (proliferating / non-proliferating cells)
Cells were grown under two different conditions – in complemented medium (e.g.
10% FBS) and in low-serum medium (0.02% FBS). A volume of 1 ml radiolabeled conjugate with an radioactivity concentration of about 23 kBq/ml was added to all dishes as described above. At the time the retention was started (after pre-incubation) the cell numbers were about 3x104 in dishes with complemented medium and 1x105 in low serum dishes. The cell dishes were incubated with conjugate for 24 hours at 37 C and were then washed six times in ice-cold serum free medium. A volume of 1 ml fresh medium was added to each dish and this retention medium was also changed after two days (52 hours). The retention was studied for five days (124 hours). At each time point, three dishes from each FBS concentration were washed and trypsinated as described above. The cells were counted, and the radioactivity was measured in a gamma counter.
Molecular weight distribution of excreted compounds was analyzed on retention medium from 52 h and 102 h as previously described. NAP-5 columns equilibrated with complemented medium and 0.02% FBS medium respectively were used.
Recharging (proliferating / non-proliferating cells)
Proliferating and non-proliferating cells were grown as above. The number of cells at the time of the first retention was about 1x105. Conjugate diluted in medium to an radioactivity concentration of about 22 kBq/ml was added to all dishes and incubation at 37 C was started. At different time points up to 24 hours triplicates of both FBS concentrations were taken. The dishes were washed six times in ice-cold serum free medium, trypsinated, and cell numbers and radioactivity were measured. The rest of the dishes were carefully washed six times and 1 ml fresh medium was added to each dish. Retention studies were conducted for 24 hours, as previously described.
Remaining dishes were washed again six times, and 1 ml conjugate diluted in medium to an radioactivity concentration of about 4 kBq/ml was added to all dishes. The binding and retention of this second “boost” was measured in the same way as the first one.
Results and Discussion
Conjugation
Preparative Gel Electrophoresis
The results from the preparative gel electrophoresis showed a large peak around fraction 20-30 see Figure 4. This peak contained EGF-dextran conjugate, as seen in Figure 5. A few other peaks were observed and further analyzed by gel filtration.
Fraction 19 was shown to contain conjugate. The other peaks contained EGF and 125I, but the signals obtained were too weak for us to be able to discriminate true peaks from artifacts. Since it was uncertain at the time whether fraction 19 contained conjugate or something else, only fractions 22-29 were pooled.
A gel filtration analysis (see Figure 5) of the conjugate later in the purification process showed that the pool contained pure conjugate. The peak at fraction 36 in Figure 5 is free 125I possibly derived from radiolysis of the conjugate.
Figure 4 Preparative Gel Electrophoresis. The electrophoresis was run at 300 V, 1 W, 3 mA (1 W kept constant). Flow rate 50 µl/min.
Fractions 22-29 were pooled.
0 100000 200000 300000 400000 500000 600000
0 10 20 30 40 50
R a d i o a c t i v i t y [ C P M ]
Fraction [ml]
Figure 5 Gel filtration of conjugate pool. Separation was made on a Superdex 75 column equilibrated with PBS. The flow rate was 1 ml/min.
0 5000 10000 15000 20000
0 10 20 30 40 50
R a d i o a c t i v i t y [ C P M ]
Fraction [ml]
Radioactivity [CPM] Radioactivity [CPM]
Proteinase K degradation
Intact conjugate and conjugate treated with Proteinase K were analyzed using gel filtration. Even though the same amount of radioactivity was added, the eluted amount differed greatly. To correct for this, the percentage CPM in each fraction was counted and plotted (Figure 6). The amount of radioactivity left in the conjugate peak after degradation (e.g. the dextran part of the conjugate) was calculated to be 57%. This result correlates well to previous experiments [13].
Cellular tests
Cell proliferation test
The results from the cell proliferation test are shown in Figure 7 and summarized in Table 1. The FBS concentration chosen for low-serum condition in the following experiments was 0.02%, even though 0% FBS seemed to be a stable condition.
Previous experiments on the proliferation rate of this cell line have shown that U-343 MGaCl2:6 cells have a cell division rate of about 36 h when cultured in 10% FBS [17]. The measurements done here are thus well correlated to previous results.
10% FBS 0.05% FBS 0.02% FBS 0% FBS
Cell division rate 35 h 105 h 594 h -
Figure 6 Proteinase K degradation separated on a Sephadex 75 column equilibrated with PBS and run at 1 ml/min. The peak around fraction 16 is conjugate and the small peak around fraction 36 is 125I. Degradation products are found in fractions in between these two peaks.
Table 1 Cell division rates calculated from curve fits of data obtained from cell proliferating test
0 5 10 15 20 25
0 10 20 30 40 50
Degraded conjugate Un-degraded conjugate
P e r c e n t a g e [
% ]
Fraction [ml]
Percentage [%]
0 200000 400000 600000 800000
0 20 40 60 80 100 120 140 160
0% FBS 0.02% FBS 0.05% FBS 10% FBS
N u m b e r o f c e l l s
Time [hours]
Conjugate binding test
Figure 8 shows the binding time pattern for the conjugate to the cells. A rapid increase in binding is observed during the first hours of incubation. A saturation effect seems to occur after about eight hours of incubation probably due to receptor saturation.
Figure 7 Cell proliferation experiment. Each point is an average of three points. Error bars indicate highest and lowest measurement
Figure 8 Conjugate binding test. Each point is an average value of three points. Error bars indicate highest and lowest measurement.
0 2000 4000 6000 8000 10000 12000
0 5 10 15 20 25 30
C e l l A s s o c i a t e d R a d i o a c t i v i t y
[ C P M ]
Time [hours]
Number of cellsCell associated radioactivity [CPM / 10^5 cells]
Displacement
The displacement test, in which unlabeled EGF competes with conjugate for binding to the cells, investigated the specific binding to EGF receptors. The results in Figure 9 shows that the conjugate binds specifically to the EGF receptor with about 5%
unspecific binding.
Retention – internalized / membrane bound
Figure 10 and 11 show the cell-associated retention of radioactivity per cell after two different pre-incubation times. More radioactivity has bound after a pre-incubation period of 24 hours, than after 1 hour, which could be expected based on the appearance of the binding curve (Figure 8). Furthermore, the results show that the level of membrane bound radioactivity is relatively constant over the 25 hour period studied especially after a long pre-incubation time. This method of discriminating between internalized and membrane bound molecules has been criticized though. Ong
Figure 9 Displacement test. Each point is an average of three. Error bars between highest and lowest measured value.
Figure 10 Conjugate retention after 1 h pre-incubation. Each point is an average of three. Error bars between highest and lowest value.
0 1000 2000 3000 4000 5000
0.001 0.01 0.1 1 10 100 1000
C e l l A s s o c i a t e d R a d i o a c t i v i
[ C P M / 1 0
^ 5 c e l l s ]
Concentration EGF [ng/ml]
0 500 1000 1500 2000 2500 3000 3500 4000
0 5 10 15 20 25
Membrane bound Internalized Total
C e l l A s s o c i a t e d R a d i o a c t i v i t y
[ C P M / 1 0
^ 5 c e l l s ]
Time [hours]
Cell associated radioactivity [CPM / 10^5 cells]
Cell associated radioactivity [CPM / 10^5 cells]
et al. [14] have shown that the low pH extraction of membrane bound molecules can extract low molecular weight catabolic products from lysosomes and also lyse cells.
The retention experiment also shows that the level of retained radioactivity is dependent on pre-incubation time. After a one hour pre-incubation the fraction left after 25 hours of further incubation is approximately 31%, whereas in the 24 hour pre- incubation case 57% remains cell associated after the same time. This correlates well with previous experiments [13].
Results of the investigations on the molecular weight distribution of the excreted substances during retention are shown in Figure 13. They show that after a short pre- incubation time, such as 1 h, mostly low molecular weight compounds are released.
Cells more saturated in conjugate uptake, as in the case with 24 h pre-incubation, show a more equal distribution between low molecular weight- and high molecular weight compounds. In the 1-h pre-incubation case, excretions from as early as 1 h after conjugate application start is collected and the LMW fraction is then high due to degradation of EGF, which mainly occurs within the first hours after incubation start [4, 16]. In the case of 24 h pre-incubation, most EGF has been degraded and excreted already during the pre-incubation period and is thus not included in the molecular weight distribution analysis.
Figure 11 Conjugate retention after 24 h pre-incubation. Each point is an average of three. Error bars between highest and lowest value.
0 5000 10000 15000 20000 25000
0 5 10 15 20 25
C e l l A s s o c i a t e d R a d i o a c t i v i t y
[ C P M / 1 0
^ 5 c e l l s ]
Time [hours]
Cell associated radioactivity [CPM / 10^5 cells]
Cellular retention of I-labelled EGF-dextran
Long time retention
The long time retention experiments were performed on both proliferating and non- proliferating cells and the results are shown in Figure 12 and 14. A comparison between these two conditions shows that about three times more radioactivity per cell bound to the proliferating cells than to the non-proliferating cells. This might be due to the fact that proliferating cells express more EGF receptors and perhaps also have a faster internalization process. The other result that could be extracted from the experiment is that the percentage of radioactivity retained in the cells after a specific time did not seem to depend on whether the cells were proliferating or not. The observed retention levels are summarized in Table 2.
Figure 12 Long time retention study of proliferating cells. Each point is an average of three. Error bars between highest and lowest value.
Figure 13 Molecular weight distribution after 24 h retention study.
0 500 1000 1500 2000 2500
0 20 40 60 80 100 120 140
R a d i o a c t i v i t y [ C P M ]
Time [hours]
0 500 1000 1500 2000 2500 3000
1 h pre-incubation 24 h pre-incubation
HMW LMW
R a d i o a c t i v i t y [ C
Radioactivity [CPM] Radioactivity [CPM ]
a b
Proliferating Non proliferating
24 hours 50% 46%
72 hours 33% 31%
124 hours 21% 20%
Table 2 Retained levels of radioactivity calculated from double exponential curves fitted from data obtained in the long time retention experiment.
The results from the molecular weight distribution analysis of the retention medium is shown in Figure 15 a and b. No relevant difference is seen between proliferating and non-proliferating cells. The distribution is the same after 52 h and 102 h, which correlates well with the hypothesis given in the introduction.
Figure 15 Molecular weight distribution of substances excreted after 52 h and 102 h respectively. In (a) the molecular weight distribution from non-proliferating cells are shown, and in (b) the molecular weight distribution from proliferating cells. Each point
Figure 14 Long time retention study of non-proliferating cells. Each point is an average of three. Error bars between highest and lowest value.
0 200 400 600 800 1000 1200
52 h 102 h
HMW LMW
R a d i o a c t i v i t y [ C P M ]
0 500 1000 1500 2000
52 h 102 h
HMW LMW
R a d i o a c t i v i t y [ C P M ] 0
500 1000 1500 2000 2500
0 20 40 60 80 100 120 140
R a d i o a c t i v i t y [
Time [hours]
Radioactivity [CPM]
Radioactivity [CPM] Radioactivity [CPM]
Recharging (proliferating / non-proliferating cells)
The results from the recharging experiment are shown in Figures 16 and 17.
Unfortunately the results are rather difficult to interpret. The variations in each point are quite large and the fact that a much lower radioactivity concentration of the conjugate was added the second time (4 kBq/ml compared to 22 kBq/ml in the first application) results in difficulties comparing the two doses.
Even though the radioactivity concentration of the second dose was very low, the retention seemed to improve markedly. A comparison between Table 3 and Table 2 shows an approximate increase in 72-h retention of about 40% by using repeated doses compared to what was to be expected if no second dose were given.
Conjugate added Conjugate added
Figure 17 Recharging experiment on non-proliferating cells. Each point is an average of three. Error bars between highest and lowest value.
Figure 16 Recharging experiment on proliferating cells. Each point is an average of three. Error bars between highest and lowest value.
Conjugate added Conjugate added
0 200 400 600 800 1000 1200 1400
0 20 40 60 80 100
R a d i o a c t i v i t y [ C P M ]
Time [hours]
0 500 1000 1500 2000 2500 3000 3500
0 20 40 60 80 100
R a d i o a c t i v i t y [ C P M ]
Time [hours]
Radioactivity [CPM]Radioactivity [CPM]
Proliferating Non proliferating
24 hours 58% 62%
72 hours 52% 57%
Table 3 Retained levels of radioactivity calculated from data obtained in the recharging experiment. Retention level after 24 h retention after the second dose (72 hours after the first dose) is called 72 h.
Conclusions
The conjugate constructed showed good binding kinetics and was shown to bind specifically to the EGF receptor. Retention studies showed that a better retention was obtained after 24 h of incubation with conjugate than after 1 h of incubation (31%
compared to 57% after 25 hours of further incubation). The amount membrane bound radioactivity was relatively constant over the 25 h period studied. The long time retention study showed that the level was quite high also after five days - almost half of the radioactivity retained after 24 hours was also retained after five days. No discrimination between proliferating and non-proliferating cells could be seen, except for the fact that proliferating cells bound more radioactivity than non-proliferating cells, possibly because they have more EGF receptors and/or a more effective internalization. The attempts to “boost” the radioactivity by the recharging experiment indicated that this could be possible, however this must be repeated before any conclusions can be made.
The results from this work show that the use of a residualizing label, such as dextran, increases the intracellular retention. With a good retention the radionuclides have a longer time to irradiate the tumor cell nucleus and thus a higher probability of killing the cell. These experiments were done with a conjugate radioactively labeled on both the EGF and dextran part. About 57% of the radioactivity was located on the dextran part of the conjugate. With labels on dextran only the retention increases further.
Retention of 80% have been measured for 24 h incubation using EGF-dextran with
125I only on the dextran [13].
Acknowledgments
I would like to show my thank my excellent supervisor Åsa Liljegren for demonstrating all practical work, showing me around at the department, answering all my questions about just everything and finally for reading the manuscript over and over again. You did a really good job as my supervisor! I would also like to thank my
“backup supervisor” Lars Gedda for coming up with solutions to problems and discussing my work and results and finally for critically reading the manuscript.
Thanks also to Mark Lubberink for helping me with all computer problems that always seems to show up whenever I am around and to Bo Stenerlöw for teaching me Kaleidagraph. I would also like to thank all the other examination workers at the department for a lot of laughs and fun in our room. Finally I would like to thank my professor and examiner Jörgen Carlsson for answering questions, reading my work and for convincing me to stay at the department as a Ph.D. student.
References
1. Agosti RM, Leuthold M, Gullick WJ, Yasargil G, Weistler OD. Expression of the epidermal growth factor receptor in astrocytic tumours is specifically associated with glioblastoma multiforme. Virchows Arch A Pathol Anat Histopathol 1992;420:321-5
2. Al-Kasspooles M, Moore JH, Orringer MB, Beer DG. Amplification and over- expression of the EGFR and erbB-2 genes in human esophageal
adenocarcinomas. Int J Cancer 1993;54:213-9
3. Andersson A, Holmberg A, Carlsson J, Carlsson J, Westermark B. Binding of epidermal growth factor-dextran conjugates to cultured glioma cells. Int J Cancer. 1991;47:439-44
4. Carpenter G, Cohen S. Epidermal Growth Factor. Ann Rev Biochem 1979;48:193-216
5. Chen YF, Pan G-Z, Hou X, Liu T-H, Chen J, Yanaihara N. Epidermal Growth Factor and Its Receptors in Human Pancreatic Carcinoma. Pancreas. 1990 May;5(3):278-83.
6. Gorgoulis V, Giatromanolaki A, Karameris A, Tsatsanis C, Aninos D, Ozanne B, Veslemes M, Jordanoglou J, Trigiduo R, Papastamatiou H, Spandidos DA.
Epidermal growth factor receptor in squamous cell lung carcinomas: an immunohistochemical and gene analysis in formalin-fixed, paraffin-embedded material. Virchows Arch A Pathol Anat Histopathol 1993;423(4):295-302 7. Haigler HT, Mawfiled FR, Willingham MC, Pastan I. Dansylcadaverine
inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells. J Biol Chem 1980 Feb 25;255(4):1239-41
8. HunterWM, Greenwood FC. Preparation of Iodine-131 Labelled Human Growth Hormone of High Specific Activity. Nature 1962 May 5;194 9. Internet: http://www.aicr.org.uk/progress/cancer1.htm 2001-02-08 10. Internet: http://www.cancerfonden.se 2001-02-08
11. Irish JC, Bernstein A. Oncogenes in Head and Neck Cancer. Laryngoscope 1993 Jan;103:42-52
12. Klijn JG, Berns PM, Schmitz PI, Schmitz PI, Foekens JA. The clinical significance of epidermal growth factor receptor (EGF-R) in human breast cancer: a rewiev on 5232 patients. Endocr Rev 1992;13:3-17
13. Liljegren Å, Blomquist E, Carlsson J. Gedda L. Cellular retention of radioactivity delivered via dextranation; Model experiments with EGF- dextran. 2001 manuscript
14. Ong GL, Mattes MJ. Limitations in the use of low pH extraction to distingue internalized from cell surface-bound radiolabeled antibody. Nucl Med Biol 2000 Aug 27(6):571-5
15. Schwenchheimer K, Huang S, Cavenee WK. EGFR gene amplification – rearrangement in human glioblastomas. Int J Cancer 1995;62:145-8
16. Sorkin A. Endocytosis and intracellular sorting of receptor tyrosine kinases.
Front Biosci 1998 July 26;3:d729-38
17. Stenerlöw B, Pettersson O-A, Essand M, Blomqvist E, Carlsson J. Irregular variations in radiation sensitivity when linear energy transfer is increased.
Radiother Oncol. 1995;36:133-42
18. Stoscheck CM, Carpenter G. Characterization of the Metabolic Turnover of Epidermal Growth Factor Receptor Protein in A-431 Cells. J Cell Physiol 1984; 120:296-302
19. Werner MH, Humphrey PA, Bigner DD, Bigner SH. Growth effects of epidermal growth factor (EGF) and a monoclonal antibody against the EGF receptor on four glioma cell lines. Acta Neuropathol (Berl) 1988;77(2):196- 201
20. Wright C, Mellon K, Johnston P, Lane DP, Harris AL, Horne CH, Neal DE.
Expression of mutant p53, c-erbB-2 and the epidermal growth factor receptor in transitional cell carcinoma of the human urinary bladder. Br J Cancer 1991 Jun;63(6):967-70
21. Zhao Q. Targeting to EGF receptors, Preparation and Experimental Analysis of End-End coupled mEGF-Dextran conjugates. Acta Universitatis
Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the faculty of Medicine. 1998;762
