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UPTEC X 01 011 ISSN 1401-2138FEB 2001

ANN-CHARLOTT STEFFEN

Cellular retention of125I-labeled EGF-dextran

Master’s degree project

Molecular Biotechnology ProgrammeUppsala University School of Engineering

UPTEC X 01 011 Date of issue 2001-02Author

Ann-Charlott Steffen

Title (English)

Cellular retention of 125I-labeled EGF-dextran

Title (Swedish)

AbstractA tumor cell specific targeting molecule was constructed as a conjugate between epidermalgrowth 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 glucosepolymer that is not degraded by mammalian cells and can thus act as an effective carriermolecule, carrying radioactive nuclides for killing the tumor cell. The conjugate was tested forbinding kinetics, specificity, internalization, retention and recharging. It was found that thebinding was specific, most radioactivity was internalized and 20% of the initial radioactivity wasleft after five days when ~60% of the radioactivity was located on the dextran part of theconjugate.

KeywordsEGF, dextran, tumor targeting, residualizing label, retention

Supervisors

Åsa LiljegrenDivision of Biomedical Radiation Sciences, Uppsala University

ExaminerJörgen Carlsson

Division of Biomedical Radiation Sciences, Uppsala University

Project name Sponsors

Language

EnglishSecurity

ISSN 1401-2138Classification

Supplementary bibliographical information Pages23

Biology Education Centre Biomedical Center Husargatan 3 UppsalaBox 592 S-75124 Uppsala Tel +46 (0)18 4710000 Fax +46 (0)18 555217

Cellular retention of 125I-labeled EGF-dextran

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 tillatt patienten återinsjuknar efter en tid. Därför används cellgifter som komplement till

kirurgi för att försöka nå de spridda cancercellerna. Både cellgifter och externstrålbehandling har den egenskapen att de attackerar alla delande celler, alltså även

friska celler. Detta leder till allvarliga sidoeffekter, vilket begränsar den dos man kange 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 skullecancern 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 avdenna målsökare kan ackumulera radioaktivitet i cellen och om den är lika effektiv

mot tillväxande och vilande celler.

Examensarbete 20 p i Molekylär bioteknikprogrammetUppsala universitet Februari 2001

Contents

INTRODUCTION............................................................................................................. 2

MATERIALS AND METHODS.......................................................................................... 6

CONJUGATION ................................................................................................................. 6Radiolabeling .............................................................................................................. 6Reductive amination...................................................................................................... 6Gel filtration ............................................................................................................... 6Preparative Gel Electrophoresis ..................................................................................... 6CDAP ........................................................................................................................ 7Proteinase K degradation .............................................................................................. 7

CELLULAR TESTS ............................................................................................................. 8Cell culture ................................................................................................................. 8Cell proliferation test .................................................................................................... 8Conjugate binding test................................................................................................... 8Displacement test ......................................................................................................... 9Retention – internalized / membrane bound ....................................................................... 9Long time retention (proliferating / non-proliferating cells).................................................10Recharging (proliferating / non-proliferating cells)............................................................10

RESULTS AND DISCUSSION ..........................................................................................11

CONJUGATION ................................................................................................................11Preparative Gel Electrophoresis ....................................................................................11Proteinase K degradation .............................................................................................12

CELLULAR TESTS ............................................................................................................12Cell proliferation test ...................................................................................................12Conjugate binding test..................................................................................................13Displacement..............................................................................................................14Retention – internalized / membrane bound ......................................................................14Long time retention......................................................................................................16Recharging (proliferating / non-proliferating cells)............................................................18

CONCLUSIONS ..............................................................................................................20

ACKNOWLEDGMENTS..................................................................................................21

REFERENCES ................................................................................................................22

Cellular retention of 125I-labelled EGF-dextran

2

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 onlytreatment 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 thattumors 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 assuccessful in curing the patients as it was at killing them. The discovery of anesthetics

shortly afterwards, however, made more advanced forms of surgery possible andremained 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 discussedabove, to remove the whole tumor and becomes impossible when metastases are

present. Chemotherapy effects all dividing cells – not just tumor cells – and the sideeffects are often quite severe. Attacking the hormone system – as is done during

hormone treatment – obviously causes unwanted side effects and this method is onlypossible 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 cellsspecifically 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 beenfound. 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 bythe 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 withantibodies, but also with their natural ligands, that are much smaller. The

disadvantage of using growth factors as targeting molecules is that in vivo degradationof the targeting agent occurs quite rapidly so the toxic substance is released from the


3

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 acarrier – 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 therapeuticalpurposes 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, bladdercancers 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 apossible 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. Atargeting molecule attached to a carrier molecule with a toxic load binds to a receptor on thetumor cell membrane and is internalized in a vesicle. Proteolytic degradation of the targetingmolecule occurs, and degradation products are excreted from the cell, while the carrier moleculeremains internalized.

Targetingmolecule

Carrier

Toxic load

Receptor

Degradedgrowthfactor

Endosome

Lysosome

Cellmembrane


4

advantage of being easily chemically modified to allow conjugation to othermolecules.

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) wasused 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 twolysines, 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 humanglioma 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. Beforethis 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 canirradiate the nucleus and the higher the probability for killing the cell.

To investigate whether the radioactivity dose after applying conjugate to itsmaximum 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, formaximizing the dose delivered to each cell.

Figure 2 Schematic representation of an ideal boosting experiment. During phase 1 thefirst dose of the conjugate is applied and accumulated in the cells. The curve is flatteneddue to receptor saturation. During phase 2 the administration of conjugate is ceased andthe cell-associated radioactivity is slowly decreasing. The third phase represents thesecond dose of conjugate, which leads to a higher level of cell-associated radioactivitydue to receptor recruitment. Phase 4 represents the same phase as phase 2 but after thesecond dose of conjugate. The red dashed line represents what would happen if nosecond dose was administrated.

(1)

(3)

(2)

Time

Cell-associatedradioactivity

(4)


5

To compare the effect of the conjugate on proliferating and non-proliferating cellsin 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 moleculesexcreted 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 afterabout one hour after conjugate binding as has been described for free EGF [4, 16] was

correct also for EGF-dextran.

Necrotic region

Non-proliferatingregion

Proliferatingregion

Figure 3 Schematic representation of a micro-metastasis showing the different regionsthat can be found.


6

Materials and methods

ConjugationRadiolabeling

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-Tyrconjugate (~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. PBSwas 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 schematicallydescribed below.

Gel filtration

When the reductive amination reaction was finished, the conjugate was separatedfrom low molecular weight compounds, such as free 125I and NaCNBH3. This was

done using microspin G-25 columns (Pharmacia) equilibrated with 80 mM Tris-HClwith 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 preparativegel 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 Dextan-CH2-NH-mEGFNaCNBH3


7

pH 8.8). The running buffer used was a Tris-Glycine buffer (25 mM Tris, 200 mMGlycine, 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. Thebuffer 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 gammacounter 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 theelectrophoresis were further analyzed by gel filtration on a Superdex 75 column

(Pharmacia, Sweden) equilibrated with PBS at a flow rate of 1 ml/min.

CDAPThe 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 takingplace 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 twominutes 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 for18 hours.

The EGF-dextran-tyrosine conjugate was then separated from reagents and excessamounts 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 degradationTo 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 withproteinase 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 waterrespectively, and were incubated at 42 C over night. The samples were separated on a

mEGF-dextran mEGF-dextran-cyanate ester

mEGF-dextran-tyrosineTyrosineCDAP

(1)

0

200

400

600

800

1000

1200

1400

48 h 96 h

Molecular weight distibution

HMW

LMW

(2)

0

200

400

600

800

1000

1200

1400

48 h 96 h

Molecular weight distibution

HMW

LMW


8

Superdex 75 column equilibrated with PBS. The flow rate used was 1 ml/min andfractions of 1 ml were collected and analyzed for radioactivity content.

Cellular testsCell culture

Human glioma U-343MGaCl2:6 with 8.6x105 EGF receptors per cell [19] werecultured as monolayers in Ham’s F-10 medium supplemented with 10% FBS (Fetal

Bovine Serum, Biochrom, Germany), L-glutamine (2 mM, Biochrom, Germany) andPEST (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 testCell proliferation rate at different concentrations of FBS in the culture medium was

measured in order to find a suitable concentration where the cells were neitherincreasing 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 perday 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 (0.5ml 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 cellcounter (Coulter Z2).

Conjugate binding testEGF-dextran-tyrosine conjugate was radiolabeled, as described above, and diluted in

complemented medium to an radioactivity concentration of about 38 kBq/ml. Culturedishes 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 incubatedfor 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 allunbound 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 forcell counting and 0.5 ml for radioactivity measurement in a gamma counter.


9

Displacement test

A 1:10 dilution series of non-radioactive mEGF in complemented medium were madefrom 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 mEGFsolutions 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 andradioactivity measured to determine the cell-associated radioactivity.

Retention – internalized / membrane bound

Culture dishes with ~1.5x105 cells were washed once with ice-cold serum freemedium 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 mediumto 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 wereused to determine the membrane bound and internalized radioactivity according to the

method described by Haigler et al [7]. They were carefully washed and treated with0.5 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 radioactivitymeasurement. 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 molecularweight 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 appliedon 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 thefractions 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.


10

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 radiolabeledconjugate 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). Ateach 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 retentionmedium 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 atthe 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 incubationat 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 ofthe 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. Thebinding and retention of this second “boost” was measured in the same way as the

first one.


11

Results and Discussion

ConjugationPreparative Gel Electrophoresis

The results from the preparative gel electrophoresis showed a large peak aroundfraction 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 peaksfrom 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 isfree 125I possibly derived from radiolysis of the conjugate.

Figure 4 Preparative Gel Electrophoresis. The electrophoresis was runat 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

Radioactivity [CPM]

Fraction [ml]

Figure 5 Gel filtration of conjugate pool. Separation was made on aSuperdex 75 column equilibrated with PBS. The flow rate was 1 ml/min.The large peak around fraction 16 is conjugate and the smaller peakaround fraction 36 is 125I.

0

5000

10000

15000

20000

0 10 20 30 40 50

Radioactivity [CPM]

Fraction [ml]

Rad

ioac

tivity

[C

PM]

Rad

ioac

tivity

[C

PM]


12

Proteinase K degradation

Intact conjugate and conjugate treated with Proteinase K were analyzed using gelfiltration. 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 countedand 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%. Thisresult correlates well to previous experiments [13].

Cellular testsCell proliferation testThe 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 followingexperiments 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 columnequilibrated with PBS and run at 1 ml/min. The peak around fraction 16 isconjugate and the small peak around fraction 36 is 125I. Degradation products arefound in fractions in between these two peaks.

Table 1 Cell division rates calculated from curve fits of data obtainedfrom cell proliferating test

0

5

10

15

20

25

0 10 20 30 40 50

Degraded conjugate

Un-degraded conjugate

Percentage [%]

Fraction [ml]

Perc

enta

ge [

%]


13

0

200000

400000

600000

800000

0 20 40 60 80 100 120 140 160

0% FBS0.02% FBS0.05% FBS10% FBS

Number of cells

Time [hours]

Conjugate binding test

Figure 8 shows the binding time pattern for the conjugate to the cells. A rapid increasein 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 threepoints. Error bars indicate highest and lowest measurement

Figure 8 Conjugate binding test. Each point is an average value of threepoints. Error bars indicate highest and lowest measurement.

0

2000

4000

6000

8000

10000

12000

0 5 10 15 20 25 30

Cell Associated Radioactivity

[CPM]

Time [hours]

Num

ber

of c

ells

Cel

l ass

ocia

ted

radi

oact

ivity

[CPM

/ 10

^5 c

ells

]


14

DisplacementThe 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 9shows 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-incubationperiod 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 thelevel of membrane bound radioactivity is relatively constant over the 25 hour period

studied especially after a long pre-incubation time. This method of discriminatingbetween internalized and membrane bound molecules has been criticized though. Ong

Figure 9 Displacement test. Each point is an average of three. Error barsbetween highest and lowest measured value.

Figure 10 Conjugate retention after 1 h pre-incubation. Each point is an averageof three. Error bars between highest and lowest value.

0

1000

2000

3000

4000

5000

0.001 0.01 0.1 1 10 100 1000


[CPM/10^5 cells]

Concentration EGF [ng/ml]

0

500

1000

1500

2000

2500

3000

3500

4000

0 5 10 15 20 25

Membrane boundInternalizedTotal


[CPM/10^5 cells]

Time [hours]

Cel

l ass

ocia

ted

radi

oact

ivity

[CPM

/ 10

^5 c

ells

]

Cel

l ass

ocia

ted

radi

oact

ivity

[CPM

/ 10

^5 c

ells

]


15

et al. [14] have shown that the low pH extraction of membrane bound molecules canextract 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 leftafter 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 wellwith 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 todegradation 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 excretedalready 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 anaverage of three. Error bars between highest and lowest value.

0

5000

10000

15000

20000

25000

0 5 10 15 20 25


[CPM/10^5 cells]

Time [hours]

Cel

l ass

ocia

ted

radi

oact

ivity

[CPM

/ 10

^5 c

ells

]


16

Long time retentionThe 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 cellbound 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 afaster internalization process. The other result that could be extracted from the

experiment is that the percentage of radioactivity retained in the cells after a specifictime 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 anaverage 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

Radioactivity [CPM]

Time [hours]

0

500

1000

1500

2000

2500

3000

1 h pre-incubation 24 h pre-incubation

HMWLMW

Radioactivity [CPM]

Rad

ioac

tivity

[C

PM]

Rad

ioac

tivity

[C

PM ]


17

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 exponentialcurves fitted from data obtained in the long time retention experiment.

The results from the molecular weight distribution analysis of the retention medium isshown 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, whichcorrelates well with the hypothesis given in the introduction.

Figure 15 Molecular weight distribution of substances excreted after 52 h and 102 hrespectively. In (a) the molecular weight distribution from non-proliferating cells areshown, and in (b) the molecular weight distribution from proliferating cells. Each pointis an average of two. Error bars between highest and lowest value.

Figure 14 Long time retention study of non-proliferating cells. Each point is anaverage of three. Error bars between highest and lowest value.

0

200

400

600

800

1000

1200

52 h 102 h

HMWLMW

Radioactivity [CPM]

0

500

1000

1500

2000

52 h 102 h

HMWLMW

Radioactivity [CPM]

0

500

1000

1500

2000

2500

0 20 40 60 80 100 120 140

Radioactivity [CPM]

Time [hours]

Rad

ioac

tivity

[C

PM]

Rad

ioac

tivity

[C

PM]

Rad

ioac

tivity

[C

PM]


18

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 pointare 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 2shows 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 addedConjugate added

Figure 17 Recharging experiment on non-proliferating cells. Each point is anaverage of three. Error bars between highest and lowest value.

Figure 16 Recharging experiment on proliferating cells. Each point is anaverage of three. Error bars between highest and lowest value.

Conjugate addedConjugate added

0

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Radioactivity [CPM]

Time [hours]

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100

Radioactivity [CPM]

Time [hours]

Rad

ioac

tivity

[C

PM]

Rad

ioac

tivity

[C

PM]


19

Proliferating Non proliferating

24 hours 58% 62%

72 hours 52% 57%

Table 3 Retained levels of radioactivity calculated from data obtained in therecharging experiment. Retention level after 24 h retention after the second dose(72 hours after the first dose) is called 72 h.


20

ConclusionsThe conjugate constructed showed good binding kinetics and was shown to bindspecifically 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 boundradioactivity 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 halfof the radioactivity retained after 24 hours was also retained after five days. No

discrimination between proliferating and non-proliferating cells could be seen, exceptfor the fact that proliferating cells bound more radioactivity than non-proliferating

cells, possibly because they have more EGF receptors and/or a more effectiveinternalization. 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 killingthe 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 dextranpart 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 with125I only on the dextran [13].


21

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 andover 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 anddiscussing my work and results and finally for critically reading the manuscript.

Thanks also to Mark Lubberink for helping me with all computer problems thatalways 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 thedepartment 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.


22

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http://www.cancerfonden.se


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