R E I G T T RADIO ELECTRO IMMUNO-GENE TUMOUR THERAPY Bertil R.R. Persson PhD, MD h.c. Professor of medical radiation physics Lund University, 221 85 LUND Sweden

Abscopal effect on subcutaneously implanted N29 rat glioma tumours, from contra-lateral treatments with pulsed electric fields, radiation therapy and immunization with syngeneic interferon-gamma secreting tumor cells.

Biomedical Centre, Lund University, Bertil R.R. Persson1,4), Catrin Bauréus Koch1,4), Gustav Grafström1,4), Crister Ceberg1,4), Bengt Widegren2,4) and Leif G. Salford3,4). Dept. Radiation Physics1), Dept of Tumor Immunology2) , Dept of Neurosurgery3) Rausing Laboratory4), Biomedical Centre, Lund University, 221 85 LUND Sweden Contact: bertil_r.persson@med.lu.se

EANO 6 May-2005 Bertil.Persson@radfys.lu.se

N29 tumours inoculated Subcutaneously on both sides 200 000 cells 50 000 cells Only the right tumour was irradiated EANO 6 May-2005 Bertil.Persson@radfys.lu.se

PEF treatment EANO 6 May-2005 Bertil.Persson@radfys.lu.se

Radiation therapy (RT) 200 000 cells Only the right tumour was Irradiated with 60Co  radiation EANO 6 May-2005 Bertil.Persson@radfys.lu.se

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) EANO 6 May-2005 Bertil.Persson@radfys.lu.se

Tumour growth rate of subcutaneous N29 tumours: Controls and after treatment with PEF, RT, IFN immunization or their combination EANO 6 May-2005 Bertil.Persson@radfys.lu.se

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 EANO 6 May-2005 Bertil.Persson@radfys.lu.se

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. EANO 6 May-2005 Bertil.Persson@radfys.lu.se

Specific therapeutic effect of subcutaneous N29 tumors after RT, Immunization with IFN and their combination RT+ IFN . EANO 6 May-2005 Bertil.Persson@radfys.lu.se

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 EANO 6 May-2005 Bertil.Persson@radfys.lu.se

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. EANO 6 May-2005 Bertil.Persson@radfys.lu.se

EANO 6 May-2005 Bertil.Persson@radfys.lu.se

Intracerebral implanted N29 tumours EANO 6 May-2005 Bertil.Persson@radfys.lu.se

The rat glioma models N29 Stereotactic implantation of 500 cells in in the brain (caudate n.) 6-7 weeks later, a tumour (diam. 4-6 mm) giving symptoms, has developed In vitro culture of rat glioma cells EANO 6 May-2005 Bertil.Persson@radfys.lu.se Salford 99

Immunization against experimentally induced brain tumour N29 brain tumour cells N29 brain tumour cells Transfected with IFN- gene IMMUNIZATION Stereotactic injection of 5000 cells Day 7 Radiation therapy Day 0 Day 7, 21, 35 By ip incection of 10 million cells Day 100 2 out of 8 dead! 6 out of 7 dead Day 100 Immuneresp EANO 6 May-2005 Bertil.Persson@radfys.lu.se

Survival diagram for the different treatments EANO 6 May-2005 Bertil.Persson@radfys.lu.se

Intracerebral implanted N32 tumours EANO 6 May-2005 Bertil.Persson@radfys.lu.se

Survival diagram for the different treatments EANO 6 May-2005 Bertil.Persson@radfys.lu.se

Change in Mean survival time EANO 6 May-2005 Bertil.Persson@radfys.lu.se

Table I. Tumour Growth rate ”TGR” of subcutaneous tumours implanted on both Right and Left hind leg. The right tumour was treated with radiation (RT). Average of all experiments Resultat Right SE N LEFT t ctrl H t Ctrl /V t H/V Controls 8.5  0.3 40 9.1  0,3 0.1 INFg 7.6  0.6 19 9.2  0,8 0.3 0.9 RT 4.5 15 6.1  0.4 10-10 10-6 0.003 RT +INFg 5.9  0.5 7 6.4 0.002 0.0004 0,49 EANO 6 May-2005 Bertil.Persson@radfys.lu.se

Number of Survivals and mean survival time of intra cerebral tumours treated with IFN cell immunization, radiation therapy and their combination. Date 2003-02-28 Num. Survival / Num. animals Median Survival time days Significance t test Controls 1/6 60 Immunization with IFN-g 2/6 147 0.08 RT 5 Gy 0/8 45 0.2 RT 15 Gy 2/8 88 0.4 RT 5 Gy + Immunization 3x 6/8 154 0.01 RT 15 Gy + Immunization 3x 5/8 118 0.1 EANO 6 May-2005 Bertil.Persson@radfys.lu.se

Log TV of Controls EANO 6 May-2005 Bertil.Persson@radfys.lu.se

Number of rats dead and alive in the various groups of treatment at 130 days after inoculation . EANO 6 May-2005 Bertil.Persson@radfys.lu.se

Mean survival time at 130 days after inoculation in the various groups of treatment. EANO 6 May-2005 Bertil.Persson@radfys.lu.se