1. Lund University, 221 85 LUND Sweden
B R I G T T
Brain RADIO IMMUNO GENE TUMOUR THERAPY
Bertil R.R. Persson
Professor of medical radiation physics
Lund University,
LUND Sweden

2. Bertil R.R. Persson, Catrin Bauréus Koch,
Therapeutic effect of radiation therapy combined with immunization by syngeneic IFN-gamma secreting tumor cells on N29 and N32 brain tumors implanted either subcutaneously on the flank or in the brain of Fischer 344 rats.
Bertil R.R. Persson, Catrin Bauréus Koch,
Gustav Grafström, Crister Ceberg, Per Engström,
Bengt Widegren and Leif G. Salford

3. Subcutaneous Tumours The rat glioma N29 was induced in The
Rausing Laboratory (BMC Lund Sweden) by subcutaneous administration in the hind legs.
cells were inoculated into
the right leg as primary tumour
cells were inoculated into the
left leg, as secondary tumour (metastasis)
Tumour volume is estimated as an ellipsoid by length, width and thickness measured with a calliper. When the tumours reached a volume
of 9 cm3, the animal was sacrificed of ethical reasons.

4. N29 tumours inoculated Subcutaneously on both sides
Only the right
tumour was
irradiated

5. Tumour growth rate (TGR) of subcutaneous N29 tumours: Controls and after treatment with RT, IFN immunization or their combination RT+ IFN

6. Specific therapeutic effect (STE) of subcutaneous N29 tumors after RT, Immunization with IFN and their combination RT+ IFN .

7. Intracerebral implanted N29 tumours
By stereotactic technique 5000 N29 cells in 5 l nutrient solution were injected with a Hamilton syringe into the head of the right caudate nucleus of Fischer 344 rats.
To avoid extra-cranial tumour growth, the injection site was cleaned with 70% ethanol after injection and the borehole was sealed with wax.

8. The rat N29 & N32 glioma models
2. Stereotactic implantation
of 5000 glioma cells in in
the brain (caudate nucleus)
a tumour (diam.
4-6 mm) giving
symptoms, has
developed
Untreated controls
N29  6-7 weeks later
N32  2-3 weeks later
1. In vitro culture
of rat glioma cells
N29 and N32
3. Treatment
7 days later

9. Inoculation of Intracerebral Tumours
By sterotaxic technique 5000 N29 or N32 cells in 5 l nutrient solution were injected with a Hamilton syringe into the head of the right caudate nucleus of Fischer 344 rats.
To avoid extra-cranial tumour growth, the injection site was cleaned with 70% ethanol after injection and the borehole was sealed with wax.

10. Immunization with IFN- gene modified syngeneic tumor cells
The transfected cells were irradiated with 137Cs -rays to 70 Gy using a Gammacell 2000 (Mølsgaard Medical, Risø, Denmark) source at a dose rate of 4.0 Gy/min with a cell density of cells/ml in serum free medium (IMDM-0).
Immunization was performed with 3 miljon tumour cells (N29) injected intraperitoneally.

11. Radiation therapy
Before radiation therapy, the animals were anesthetized with Ketalar/Rompun, 0.55 ml/ 100g. Animals were given a single radiation treatment using a 60Co radiotherapy unit (Siemens Gammatron S). The radiation field size was collimated to cover the brain ( 1x1 cm2). The adsorbed dose of either 5 or 15 Gy was measured using a TLD chip placed next to the tumour in the field under the bolus.
Within one hour after the radiotherapy session the animals were immunized by given intraperitoneally injections of radiation sterelized, gene modified N29 or N32 tumour cells, secreting interferon-gamma (IFN-). The immunization was then repeated weekly for at most two more times,

12. BRIGTT against experimentally induced brain tumour
N29 or N32 brain tumour cells
Syngeneic tumour cells
Transfected with IFN- gene
Day 7
Radiation therapy
+ immunization
Day (7) 21, 35
Stereotactic
injection of 5000 cells
Day 0
IMMUNIZATION
By ip injection
of 10 million cells
N29
Day 400
N29
Day 400
N32
Day 40
All dead
BRIGTT
2 out of 8
dead!
Controls
6 out of 7
dead
Immuneresp

13. Survival diagram for the different treatments of N29 tumors

14. Number of Survivals and mean survival time of intra cerebral tumours treated with IFNg cell immunization, radiation therapy and heir combination.
Date
N29 tumors
Num. Surv
> 400 d /
Num. animals
Significance
Survivals
> 400 d
vs Ctrl
Median
Survival time days
Controls
1/6
60  142
Immunization with IFN-g
2/6 *
405  166
RT 5 Gy
0/8 NS
45  14
RT 15 Gy
2/8
88  150
RT 5 Gy + Immunization 3x
6/8
***
412  140
RT 15 Gy + Immunization 3x
5/8
252  167

15. Challenge-study
The 14 out of 48 animals with N29 tumors that survived 400 days and 4 new controls (C1) were inoculated with 200 000 N29 glioma cells in 200 l just under the skin in the thigh of the hind leg of the rat.

16. Challenge of N29 survivals

17. Intracerebral implanted N32 tumours Exp: 2003-03-25 & 2003-09-08

18. Survival diagram for the different treatments of N32 tumors

19. Survival diagram for the different treatments of N32 tumors

20. Conclusions tumors in rat with single session of radiation therapy
Combined treatment of intracerebral implanted glioma
tumors in rat with single session of radiation therapy
treatment combined with immunization by syngeneic
interferon-gamma (IFN-) secreting tumor cells (BRIGTT)
increase the survival time significantly.
BRIGTT treatment of slowly growing N29 glioma in 8 rats
resulted in 6 complete remissions (75%; >400 d).
Challenge with tumour cell injection on the flank of the
surviving animals at 400 days after the first implantation
resulted in tumour growth in 50 % of the animals.
BRIGTT treatment of 12 animals with rapidly growing N32
tumors resulted in no complete remissions

22. The aim of the study
The aim of the present study is to investigate the therapeutic effects of immunization with interferon-gamma secreting cells in combination with radiation therapy in a rat model with N29 or N32 rat glioma tumours.

23. Radiation theraphy (RT)
Only the right
tumour was
Irradiated with
60Co  radiation

24. An exponential tumour growth model is applied to quantitatively evaluate and compare the effect of tumour treatment in the various experimental groups on the tumour growth

25. Tumour Volume of untreated Controls in 4 different experimental series

26. Immunized with IFNg secreting cells
Experiment IFNg
Experiment IFNg

27. Radiation therapy of the right tumour with 4 fractions of 5 Gy (total 20 Gy)
Experiment RT
Experiment RT

28. Combined treatment with radiation therapy of the right tumour with 4 fractions of 5 Gy (total 20 Gy) and immunization with IFNg secreting

29. Exponential Tumour Growth Model
Tumour growth rate “TGR” is estimated from the tumour volume measurements by fitting the data of each individual tumour to a model of exponential growth
where
“TVt” is Tumour volume at time t
t is time after first treatment.
“TV0” is Tumour volume at time t = 0,
“TGR” is tumour growth rate constant (% per day)

30. Only the right tumour was irradiated (RT).
Tumour Growth rate ”TGR” of subcutaneous N29 tumours implanted on both Right and Left hind leg.
Only the right tumour was irradiated (RT).
Result
Right SE N
LEFT
t ctrl H
t Ctrl /V
t H/V
Controls
7.8
 1.5 24
8.4
0.1
INF
6.9
 0.9 7
8.1
 1.0
0.2
0.3
0.4 RT
4.5
 0.3 15
6.1
 0.4
10-9
10-4
0.003
RT + INF
5.9
 0.5
6.4
0.02
0.004
0,49
Average of experiments and

31. The therapeutic effect “TE = -lnS” is defined as the ratio between the tumour volume of the treated tumour and the control
where TVE Tumour volume of the Exposed group TVC Tumour volume of the Control group S Tumour “Surviving fraction” TGRE Tumour growth rate constant of the exposed group day-1 TGRC Tumour growth rate constant of the control day-1 S Surviving fraction of the tumour at t days after treatment was started

32. The tumour growth rate TGR of the controls evaluated at various time intervals after inoculation
Experiment
Right tumour
Left tumour
TGR
% per day SD
Num
Num.
CTRL H7-20
10,6
3.4 51
CTRL V7-20
20.6
7.3
CTRL H7-30
9.9
3.7 23
CTRL V7-30
14.0
4.7 22
CTRL H>7
8.0
1.8
CTRL V>7
9.6
2.0 18
CTRL H>20
7.0
CTRL V>20
2.8 17
CTRL H>30
6,9 15
CTRL V>30
8.2
1.9 11
Experiment
Right tumour
Left tumour
TGR
% per day SD
Num.
CTRL H >26
9.0
1.9 7
CTRL V>26
8.6
0.7
CTRL H > 32
8.3
0.8
CTRL V > 32
8.0
CTRL H > 40
7.2
0.9
CTRL V > 40
6.7
0.5

33. The therapeutic effect “TE”:
TE = (TGRc-TGRE) • t
Depends on time and the characteristics
of each experiment
By dividing with TGRC•t we get a parameter
invariant with time and experimental conditions

34. Specific Therapeutic Effect “STE” is defined as follow.
The average of the individual Tumour growth rate constant in the group of exposed rats. day-1
The average of the individual Tumour growth rate constant in the group of control rats. day-1

35. The STE is equal to 0 when the average of tumour growth rate constant of the exposed group, is equal to the average of the tumour growth rate constant of the control.
The STE is equal to 1 when the average tumour growth rate constant of the exposed group, is equal to 0.

36. Specific Therapeutic Effect “STE” of subcutaneous N29 tumours implanted on both Right and Left hind leg.
Exp. & Date
IFNg
STE Right sd N
STE Left E
0.08
 0.34 2
-0.15
 0.19 D
0.26
 0.03 8
0.14
 0.01 C
-0.10
 0.02
0.02
 0.002 B
-0.76
 0.18 5
-0.11
 0.15 A
-0.01
 0.001 3
0.03
Average All
 0.17 26
 0.05
IFNg B;E
-0.34
 0.08 7
-0.13
 0.07 RT
Right
Left
0.35
0.18
0.45
0.05
 0.04
RT B;E
0.40 15
0.12
 0.06
RT+INFg
0.37
RT+INFg B

37. Synergetic effects of combined treatment
It is if great interest to know if there is a therapeutic gain by combining radiation therapy and immunization with syngeneic IFN secreting tumour cells

38. Therapeutic Enhancement Ratio.
“TER” of the combined treatments is the ratio of the specific therapeutic effect “STE” of the experimental combination of Electrical pulses and radiation and the hypothetical combination of the two agents used independently
where the hypothetical specific therapeutic effect by independent (additive) action of ionizing radiation and Electrical pulses is given by

39. Therapeutic Enhancement Ratio
“TER” is a measure of any synergistic or diminishing effect obtained in the combination of the two agents. TER > 1 may due to synergistic interaction of sub lethal lesions induced by both agents to produce more lethal events.

40. Therapeutic Enhancement Ratio “TER” of subcutaneous tumours implanted on both Right and Left hind leg. The right tumour was treated with radiation (RT).
Therapeutic Enhancement Ratio:
Right Sd
Left B
-1,2
-0,7
-1,3
-3,5
B /B, E
6,2
9,7
-8,0
-73,9
B /All & BE
1,3
0,8
0,7
0,6

41. Intracerebral implanted N29 tumours Exp; 2002-09-23

42. The rat glioma models N29 Stereotactic implantation in
in the brain (caudate n.)
Without treatment
6-7 weeks later,
a tumour (diam.
4-6 mm) giving
symptoms, has
developed
In vitro culture
of rat glioma cells
treatment
7 days later
Salford 99

43. Survival diagram for the different treatments

44. Number of survivals and dead rats at 130 days after inoculation in the various groups of treatment.

45. Mean survival time at 130 days after inoculation in the various groups of treatment.

46. Tumour Weigth

48. Survival diagram for the different treatments

49. Median survival time

50. Change of Median survival