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intravenously (iv ) via the tail vein the dose for each experiment is noted in the

results sections. Groups of four animals (unless otherwise stated) at various time intervals were anaesthetised and bled by the retro-orbital venous sinus. The mice were immediately killed after this the tumour and various normal tissue samples were excised. The tissues were blotted to remove surface blood, placed

in pre-weighed tubes and the weights recorded before digesting overnight in 2

ml (approx) 7M KOH. A sample of injected antibody (1 /1 0 of the administered dose) diluted in 2 ml KOH w as counted using a gamma counter at the same time

as the digested tissues to allow results to be expressed as a percentage of injected activity per gram of tissue. There w as no correction for radioactive decay for biodistribution experiments.

*(NB. An experiment w as performed to assess the difference in biodistribution of radiolabelled antibody in TO and MFl m ouse strains. Results demonstrated <2% difference in normal tissue distribution, clearance and tumour uptake of

radioconjugate in tumour bearing TO and MFl mice.)

2.11.2.1 Statistical analysis

Due to the small number of mice per group statistical analysis w as

performed, where appropriate, to compare two individual data sets in attempt to indicate whether they were significantly different. The non-parametric Mann- W hitney test w as selected, and the p value of 0.05 w as chosen as the critical level of significance.

2.11.3 Radioimmunoscintigraphy

Mice w ere anaesthetised with subcutaneous injection of 100 pi a 1 /1 0 dilution in saline of hypnodil (Janssen Animal Health, Oxford, UK), further injections were performed if necessary. Mice were scanned for 10 m in using a gamma-camera (IGF Gemini) fitted with a pinhole collimator. Scanning w as performed by Dr A. Green in the Dept, of Clinical Oncology.

2.11.4 Radioimmunotherapy experiments

Experiments were carried out w hen the tumours were between 0.1 0.2 cm 3 in volum e and in exponential growth usually 10-14 days after

passaging. Six mice were used per group and 6 untreated mice were used

as a control group. Radiolabelled antibody w as injected iv via the tail vein.

Tumours were measured and the mice w eighed on the day of injection and every 3 - 4 days until the tumour volum e reached 2 cm^, w hen the mice w ere sacrificed.

Calculation

Tumour volum e (cm^) = (Length (cm) x Width (cm) x Depth (cm)

x T z )

Standard error of the mean (s.e.m) = Standard D eviation/ V»

where n = number of mice

2.11.4.1 White Cell Counts

For an assessment of toxicity w hole white cell counts (WBC) were measured in 4 mice per group and in 4 untreated mice prior to injection and at w eekly intervals until the WBC returned to normal or mice were sacrificed. Blood from anaesthetised mice (25 pi) was diluted 1/2 0 in 475 pi diluting fluid

(2% acetic acid coloured pale violet with a few drops of crystal gentian violet).

The diluting fluid lysed the red cells leaving white cells intact with their nuclei stained blue/black. The cells were counted using a haemocytometer (Improved Neubauer, Weber Scientific, UK).

Calculation

white cells / ml blood = Average number of cells per mm^ x 10^ x 20

2.12 D o sim e try and clearance

2.12.1 Xenograft studies

Figures for % of injected activity per g (% ia / g) for each tissue for

individual mice were entered into the dosimetry spreadsheet (Microsoft Excel), and the average median value was calculated for each time point. These values were then decay corrected using the decay correction equation:

A=Aoe"^^ where L n2/t 1/2-

t = time after injection

Ao = original activity at time t

A = activity at time t after decay correction

t i/2 = half-life of 194 h for 1^1% and 64 h for

The area under the % ia /g over time (h) curve (AUC) w as calculated using the trapezoidal rule assum ing no activity in the tumour at time 0 and 40% in the blood at time 0. This value for blood w as generated from an experiment w ith

both 1311 labelled A5B7 IgG and F(ab')2 , in which blood samples were counted at

30 seconds post injection. An average of 39% IgG and 40.7% F(ab')2 % i a / g was

present in the blood at this early time point. This is consistent w ith the estimated total blood volum e of a 20-30g m ouse being in the range 2.0-2.5 ml as described

by Durbin et a l, (1992).

To calculate total p dose to individual organs the MIRD S factor of 0.369 for 131% and 1.93 for ^Oy (MIRD Pamphlet 11,1975) w as used to convert M B q /g to cG y/h . This calculation does not take into account contribution of y energy as m ost of this penetrating radiation escapes the m ouse and there is virtually no self-absorption of y rays . Also assumes that there is uniform distribution of activity, and there were no cross-organ p radiation doses

2.12.2 Patient studies

Blood samples were taken at regular time intervals and the activity of w eighed samples was measured using a gamma counter. Antibody clearance from the circulation was estimated by calculating the % ia /k g for w hole blood samples over time and fitting this data to a bi-exponential equation and

Ct = Co (ae "^1* + be )

using the constraint (a + b) = 1

where Ct = % ia /k g at time t and Cq = % ia /k g at time 0

X= L n 2/ti/2

The m odel uses the least squares system the achieve the best fit to the data. This bi-exponential m odel of blood clearance was used to calculate a and p half-lives using the follow ing equations:

a 1/2 = Ln (2)/ Pl/2 = Ln (2)/ X2

Activity in other organs was determined by gamma camera imaging by w hole body planar and thorax and serial abdominal single photon em ission computerised tomographic (SPECT) scans over a period of days. This was performed by Dr A. Green and colleagues in the Dept of Clinical Oncology. Tissue activity expressed as a % injected activity kg"l for tumour, liver, kidney, lung and heart w as estimated from the SPECT images reconstructed using ICE filtered backprojection software, and corrected for decay, photon attenuation and

Compton scatter (Green et al., 1990). Analysis was performed by selecting

individual 0.88 cm^ regions of interest (ROI) taken from SPECT images (Lane et

al., 1994). This procedure was performed by Dr M. Napier and Miss C. Johnson

(Dept, of Clinical Oncology) with the aid of corresponding CT images. These values were corrected for P dose radiation (cGy) to each tissue using the S factor

for 1 (MIRD pamphlet no 11,1975), and the MIRD absorbed dose w eighting

system (ICRP 60,1990) which corrects for cross-organ doses.

2.13 M acro and m icro d istrib u tio n o f a n tib o d ies in tissu e sectio n s

Cryostat tissue sections were prepared and the procedures below were supervised by Mr. G. Boxer in the Dept, of Clinical Oncology.

2.13.1 Immunohistochemical characterisation of antibodies

Cryostat sections 5-7 pm thickness were prepared from frozen (stored at -70 ^C) or fresh tissue (snap frozen in iso-pentane cooled in liquid nitrogen) on a cryostat (Bright Instruments, Huntingdon, UK). Sections were mounted on 3- aminopropyl triethoxysilane (APS) coated slides, air dried and fixed in cold acetone for 20 min. The sections were blocked to minimise non-specific binding

of antibodies to tissues, by applying a blocking agent usually 1 0% normal horse

serum (Vector Laboratories, Peterborough, UK) for 10 min. The excess w as drained off and the sections incubated w ith humanised hTFM, hDFM and

hF(ab')2 (or other test antibodies) at a concentration of 2 0p g /m l diluted in tris

buffered saline pH 7.6 (TBS) for 40 min. After three 5 min w ashes in TBS the second antibody 1C2 (anti-macrocycle antibody, 20 p g /m l), w as added for 40

min, then w ashed as above. For detection sheep anti-mouse HRP (1/75, Amersham) w as applied to sections for 1 h.