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, 221 85 LUND Sweden

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

Subcutaneous Tumours The rat glioma N29 was induced in The Rausing Laboratory (BMC Lund Sweden) by subcutaneous administration in the hind legs. 200 000 cells were inoculated into the right leg as primary tumour 50 000 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.

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

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

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

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.

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

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.

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 20 000 cells/ml in serum free medium (IMDM-0). Immunization was performed with 3 miljon tumour cells (N29) injected intraperitoneally.

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,

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

Survival diagram for the different treatments of N29 tumors

Number of Survivals and mean survival time of intra cerebral tumours treated with IFNg cell immunization, radiation therapy and heir combination. Date 2003-02-28 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

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.

Challenge of N29 survivals

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

Survival diagram for the different treatments of N32 tumors

Survival diagram for the different treatments of N32 tumors

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

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.

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

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

Tumour Volume of untreated Controls in 4 different experimental series

Immunized with IFNg secreting cells Experiment 020404 IFNg Experiment 010529 IFNg

Radiation therapy of the right tumour with 4 fractions of 5 Gy (total 20 Gy) Experiment 020404 RT Experiment 010529 RT

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

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)

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 010529 and 020404

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

The tumour growth rate TGR of the controls evaluated at various time intervals after inoculation Experiment 010529 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 020404 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

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

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

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.

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 020404 0.08  0.34 2 -0.15  0.19 D 020102 0.26  0.03 8 0.14  0.01 C 010827 -0.10  0.02 0.02  0.002 B 010529 -0.76  0.18 5 -0.11  0.15 A 981112 -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

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

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

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.

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 010529 -1,2 -0,7 -1,3 -3,5 B 010529/B, E 020404 6,2 9,7 -8,0 -73,9 B 010529/All & BE 1,3 0,8 0,7 0,6

Intracerebral implanted N29 tumours Exp; 2002-09-23

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

Survival diagram for the different treatments

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

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

Tumour Weigth

Survival diagram for the different treatments

Median survival time

Change of Median survival